1 Introduction

Dendritic cells (DCs) were identified in mouse spleen in the 1970s by Ralph Steinman [1]. They are bone marrow-derived cells that are found at low frequency in all tissues and are indispensable to the immune system. DCs have the ability to recognize bacteria, virus, fungi, and other immunogenic agents via numerous pattern recognition receptors , taking up and processing the antigen into peptide form and presenting it to naive T cells in order to initiate an antigen-specific immune response [2]. Mice deficient in DC are incapable of mounting an immune response to pathogens [3–5]. DCs also play a pivotal role in controlling reactivity to self-antigen in the absence of pathogens or inflammation. They maintain self-tolerance in the thymus by presenting self-antigen and either deleting developing self-reactive T cells or rendering them unresponsive and in the periphery deleting mature T cells [6, 7] and inducing the expansion of regulatory T cells [8].

By the 1990s, it was evident that murine lymphoid organ DC could be divided into two subsets based on expression of CD8α [9, 10]. These two subsets were subsequently shown to have different immune functions and were the first indication of the vast functional diversity of DC [11]. Spleen DCs are a heterogeneous group which differ in the antigen processing and presenting pathways they use and the role they play in immunity. The surface phenotype and abilities of DC vary between subtypes [12, 13] and change as they undergo the process of maturation [14].

The vast majority of splenic DCs are in an immature state as indicated by their low-level expression of major histocompatibility complex class II (MHCII) molecules and co-stimulatory molecules. In this state they are extremely efficient at mediating endocytosis and processing of antigen and its traffic to the cell surface for presentation to T cells. Upon exposure to microbial patterns or other “danger” signals, DCs undergo a process of maturation in which they cease uptake and processing of antigen. MHC and various membrane-associated co-stimulatory molecules are up-regulated, as is the production of many cytokines and chemokines. In this form they are able to induce an effector T-cell response [15].

All mature DCs are very efficient at presenting endogenous antigen; however, presentation of exogenous antigen is largely restricted to MHC class II molecules [16]. Cross presentation is a specialized function which allows particular DC subsets to present exogenous antigen via MHC class I [17] in a role which is vital to antiviral and antitumor T-cell responses and in inducing and maintaining tolerance [18].

Due to the vast heterogeneity of the DC network, our aim when devising an isolation and purification procedure was not only to ensure that DC from lymphoid tissue could be adequately and efficiently extracted and purified regardless of their maturation state, surface phenotype, and function, but to do so in a way that conserved their in vivo state. Accidental induction of DC maturation gives an inaccurate view of their in vivo steady-state form.

Early protocols depended on overnight culture at 37 °C to separate transiently adherent splenic DC from adherent macrophages . This inadvertently induced a maturation process resulting in isolated DCs that were no longer equivalent to their in vivo steady-state form, but rather resembled mature DC [14]. Subsequently even short periods at 37 °C were shown to induce maturation of DC, particularly under conditions of high DC concentration, due to these very rare cells coming into close proximity of each other [19].

We designed our isolation procedure with this limitation in mind. Our collagenase digestion of tissue is a mild treatment performed for a relatively short time at room temperature, and has been shown not to activate DC based on expression of MHCII and co-stimulatory molecules, which remain at similar levels to those found on the small number of DC that can be isolated without collagenase at 4 °C [19]. The digestion extracts all DC subtypes without any observed bias. After the digestion, DCs constitute less than 1 % of the extracted splenocytes. A short treatment with EDTA ensures that all multicellular complexes between DC and T cells are dissociated, before the first enrichment step is performed. Selection for light-density cells is the first DC enrichment step. It results in the removal of erythrocytes and dead cells as well as many other contaminating lineages. DCs account for 10–15 % of light-density cells, and it is possible to isolate them directly at this stage using either flow cytometric cell sorting or positive selection using immunomagnetic beads. However, it is more economical to enrich further via negative selection. Non-DC lineage cells are coated with a cocktail of monoclonal antibodies and depleted with anti-immunoglobulin coated immunomagnetic beads. Care must be taken when selecting the monoclonal antibodies included in the cocktail, in order to avoid losing DC subtypes that bear molecules found more commonly on T cells, B cells, and macrophages . Residual contaminating autofluorescent macrophages and NK cells are often a problem [20, 21]. Ideally, inclusion of monoclonal antibodies specific for these cells in the depletion cocktail would be the preferred solution. Unfortunately, the candidate macrophage molecules that could potentially be targeted are also expressed on some DC subtypes and so cannot be used, and we have not been able to find an effective NK cell-specific monoclonal antibody that is also a rat IgG. Nevertheless, both can be removed during fluorescence activated cell sorting or analysis by gating out autofluorescent cells and CD49b+ cells, respectively. Alternatively NK cells may be removed by a second negative selection step. After depletion, spleen DCs account for approximately 90 % of the recovered cells (see Fig. 1) and are a mixture of migratory plasmacytoid DC (pDC) and lymphoid resident conventional (or classical) DC (cDC). These two subtypes are easily distinguished and separated using multicolor immunofluorescent staining and flow cytometry .

Fig. 1
figure 1

Sequential enrichment of spleen cDC. The proportion of cDC is increased from approximately 2 % of total leucocytes after the digestion up to approximately 90 % of all cells after depletion of non-DC lineage cells. cDCs are identified as CD11chigh cells

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In the steady state, mouse pDCs are produced in the bone marrow and migrate to lymphoid organs including the spleen, via the blood . They are characterized by low expression of the integrin CD11c and high expression of either of the pDC-specific molecules siglec H or, in the steady state, CD317 and are enriched during DC isolation if the appropriate cocktail of monoclonal antibodies is used in the depletion. CD45R and CD45RA have been used in conjunction with CD11c to identify pDC in the past, but this may be problematic if no additional NK cell depletion is planned, as some NK cells express significant levels of these molecules and may inadvertently be included in any pDC gate that is set (see Fig. 2). pDCs account for approximately 15 % of DC in mouse spleen and express high levels of toll-like receptors 7 and 9, which when ligated by viral antigens lead to production of high levels of IFN-α and IFN-λ; upregulation of MHC class II, CD40, CD69, CD80, and CD86; acquisition of typical DC morphology; and the ability to present foreign antigen [22].

Fig. 2
figure 2

NK cell contamination of isolated pDC. (a) cDCs are identified as CD11chigh and negative for either of CD45R, CD45RA, siglec H, or CD317, while pDCs are CD11clow and positive for either of CD45R, CD45RA, siglec H, or CD317. (b) NK cells, identified as CD49b+, are significant contaminants of pDC preparations separated using CD45R or CD45RA, contributing up to 19 % of recovered cells. NK cells may be gated out during flow cytometric sorting or analysis, or by depleting using a combination of anti-CD49b conjugated to biotin and anti-biotin-coated magnetic beads. Siglec H and CD317 are better alternatives for separating pDC from NK cells with very low NK cell contamination detected in pDC preparations isolated using either

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cDCs populate most lymphoid and nonlymphoid tissue. All cDCs in the spleen are tissue resident and have developed in the spleen from a blood -borne precursor [23]. They are found in the marginal zone where they constantly acquire tissue and blood antigen. Spleen cDCs are distinguished from, and can be separated from, pDC by higher expression of CD11c and the absence of siglec H, CD317, CD45R, and CD45RA [24].

Early observation of CD8 on the surface of spleen cDC made it possible to begin dividing spleen cDC into two subsets: CD8+ and CD8−. Although CD8 was adequate for cDC identification in mouse lymphoid tissue, it did not translate well to use in cDC in nonlymphoid tissue or in humans. The introduction of new markers provides a more complete characterization of the two populations. The CD8+ cDC can be defined as CD8+CD11b−CD24highCD205+CD4−CD172alowClec9A+, while the CD8− cDC are CD8−CD11b+CD24lowCD205−CD4−/+CD172ahighClec9A− [25] (see Fig. 3a). Current terminology refers to these two populations as CD8+ and CD11b+ cDC.

Fig. 3
figure 3

Separation of mouse spleen cDC into discrete subtypes. (a) Traditionally cDCs have been separated using CD8 and CD4, but alignment of spleen cDC using neither CD8 or CD4, is best accomplished using CD24 and either CD11b or CD172a. The CD24highCD11b−CD172alow and the CD24lowCD11b+CD172ahigh populations correspond to the CD8+CD4− and CD8−CD4+ subtypes, respectively. (b) Clec9A can also be used to distinguish the traditional CD8+ and CD8− cDC. Clec9A+ cDCs are CD8+CD172alow, and Clec9A− cDCs are CD8−CD172ahigh. (c) The CD8− cDC subtype can be divided further using CD4, ESAM, and Clec12A. The traditional CD4+CD8− cDCs are ESAM+Clec12Alow, and the CD4−CD8− cDCs are largely ESAM−Clec12A+

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CD8+ cDCs account for approximately 25 % of mouse spleen DC, respond to TLR3 stimulation [26], secrete IL-12p70 [27] and IFN-λ [28], and are essential for cross-presenting antigen [29, 30]. Expression of Clec9A on CD8+ cDC is of great importance (see Fig. 3b). Clec9A is a receptor for necrotic tissue which binds and directs tissue antigens associated with this tissue to the cross-presentation pathway [31, 32]. Further, Clec9A allows us to align the CD8+ cDC subtype in mouse with its functional human equivalent, which does not express CD8 [33]. Terminal differentiation of CD8+ cDC is dependent on GM-CSF and results in expression of CD103 and the acquisition of cross-presentation functions [34, 35].

The CD11b+ cDCs, which account for up to 60 % of DC in the mouse spleen, are a heterogeneous population and less well characterized. They may be further divided into two populations using CD4, the endothelial cell-selective adhesion molecule (ESAM) [36] and the C-type lectin Clec12A (see Fig. 3c). The CD11b+CD4+ subtype is Clec12A− and largely ESAM+ and is essential for presentation of MHCII-antigen complexes to CD4+ T cells [37]. The CD11b+CD4− subtype is Clec12A+ and ESAM− and is the superior producer of inflammatory cytokines such as CCL3, CCL4, and CCL5 after TLR engagement [38].

The rarity of splenic DC and the labor-intensive process required to isolate them in sufficient numbers have limited their availability for study. The cytokine fms-like tyrosine kinase 3 ligand (Flt-3L) and its receptor (Flt-3) play an essential role in the commitment of hematopoietic precursors to the DC lineage and their subsequent development [39]. Flt-3L is ubiquitously produced by multiple tissue stroma, endothelial cells, and activated T cells and drives DC differentiation from both mouse and human precursors [40]. Mice deficient in Flt-3 and Flt-3L have reduced numbers of both DC precursors and lymphoid tissue pDC and cDC [41–43].

Administration of Flt-3L to mice acts on the Flt-3+ fraction of bone marrow precursors [44] to produce large numbers of the equivalents of the steady-state mouse DC populations either in vivo or in vitro.

In vivo methods involve the administration of daily intravenous Flt-3L injections for a period of 10 days or subcutaneous injection of the Flt-3L secreting melanoma B16FLT3L. In both cases, total spleen cellularity increases up to threefold, but spleen DC numbers can be boosted up to 20-fold. DC subtypes in the spleen of mice treated in this way are identified and separated using the same markers used in spleen from untreated mice; however, there is a larger expansion of the CD8+ subtype [45, 46].

Large numbers of pDC and cDC can also be produced in in vitro cultures of bone marrow treated with Flt-3L [47, 48]. cDCs generated in these cultures do not express CD8 or CD4, so these markers are inadequate for alignment of the culture-generated cDC with the subtypes found in vivo. Equivalents of the CD8+ and CD11b+ cDC can be identified using the markers CD11b, CD24, CD172a, and Clec9A. The numbers of DC generated in these cultures can be boosted further by an initial addition of a small amount of GM-CSF. Addition of a second larger dose of GM-CSF after 6 days of culture preferentially expands the CD103+ cDC population which is the population in these cultures capable of cross presentation [34] (see Fig. 4).

Fig. 4
figure 4

Generation, separation, and modification of cDC in Flt-3L-supplemented bone marrow cultures. (a) cDCs produced in Flt-3L-supplemented bone marrow cultures do not express CD8 or CD4 but may be separated into CD8+CD4− and CD8−CD4+ equivalents using a combination of other molecules, usually CD24 or Clec9A and CD11b or CD172a. (b) Flt-3L-generated cDCs are modified by the addition of GM-CSF on day 6 of culture resulting in an increase in both the total number of cells produced and the proportion of CD103+ cross-presenting cDC after 9 days of culture. (c) Addition of GM-CSF at day 0, day 6, or both has an effect on numbers and the phenotype of DC recovered after 9 days of culture. (i) Numbers of cDC are increased two- to threefold by addition of GM-CSF at day 0 or day 6, but only marginally more if both doses are administered. (ii) pDC numbers are increased by addition of GM-CSF at day 0 but not at day 6. pDC production is slightly inhibited if GM-CSF is added on both days. (iii) The number of CD103+ cDC is increased fivefold by the addition of GM-CSF at day 6. (iv) A similar pattern of results is obtained when analysis is performed after 8 days of culture, but with a lower recovery of total DC (not shown) and CD103+ cDC

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Subpopulations of highly purified cDCs have traditionally been obtained by flow cytometric sorting. Sorting, however, is both expensive and time consuming. An alternative method, resulting in the isolation of highly purified cDC subtypes, involves positive selection using anti-fluorochrome-conjugated magnetic beads. DCs of one subtype are stained with a specific fluorochrome-conjugated monoclonal antibody, allowed to bind to anti-fluorochrome coated magnetic beads, and then selected using a magnet. If the other subtype is also required, the negative fraction may be stained with a monoclonal antibody, conjugated to another fluorochrome, that is specific for this subtype and the positive selection process repeated. In this way, it is possible to sequentially select the two cDC subtypes [49, 50] (see Fig. 5).

Fig. 5
figure 5

The sequential separation of CD8+ and CD8− cDC using fluorochrome-conjugated antibodies and anti-fluorochrome immunomagnetic beads. (a) Two major subsets of spleen DC can be visualized using the markers CD8 and CD11b: CD8+CD11b− and CD8−CD11b+. (b) The first round of selection for CD8+ DC results in a highly purified CD8+CD11b− population. (c) The flow through contains a greatly reduced proportion of CD8+ cells. (d) The flow through contains a mixture of CD11b+CD24low and CD11b−CD24hi cells. (e) A second round of depletion targets CD24 and removes many of the remaining CD8+CD24highCD11b− but also CD24highCD8− precursors of the CD8+ cDC lineage. The second flow through contains an enriched CD8−CD24lowCD11b+ population. (f) The final round of selection utilizes CD11b and results in a greatly enriched CD8−CD24lowCD11b+ population

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A similar process may be used to remove contaminating NK cells. Biotin-conjugated anti-CD49b and anti-biotin magnetic beads can be used in combination to remove and discard NK cells. Alternatively, NK cells may be removed during flow cytometric sorting.

The ability to isolate purified DC populations in an immature state and unaffected by the isolation procedure, the ability to expand their numbers using Flt-3L, and the design of economical methods to purify their subtypes , are all crucial in the ongoing study of DC function.

2 Materials

2.1 In Vivo Administration of Flt3L

  1. 1.

    Donor mice.

  2. 2.

    27G needle.

  3. 3.

    MTPBS: Mouse tonicity (308 mOsm/kg) phosphate-buffered saline. Filter through a 0.2 μM filter unit to sterilize and store at 4 °C.

  4. 4.

    Flt3L: Recombinant murine FLAG-tagged fms-like tyrosine kinase 3 ligand (constructs provided by N. Nicola, WEHI, Australia) was expressed in FreeStyle 293F cells (Invitrogen, Victoria, Australia) by transient transfection using Freestyle Max (Invitrogen) and cultured in protein-free/serum-free media (FreeStyle Expression Media, Invitrogen) for 5 days. Media containing the secreted recombinant protein was concentrated using a 10,000 molecular weight cutoff centrifugal device (Millipore, Billerica, MA). Recombinant Flt3L-FLAG was purified by affinity chromatography using an anti-FLAG M2 agarose resin (Sigma, Castle Hill, Australia) and elution with 100 mg/ml FLAG peptide (Auspep, Victoria, Australia) and further purified by size-exclusion chromatography using a prepacked Superdex 200 column (GE Healthcare, Rydalmere, Australia) (see Note 1 ).

  5. 5.

    MSA: Mouse serum albumin (Sigma Aldrich). Dissolve in MTPBS to give a 10 μg/ml stock solution. Filter through a 0.2 μM syringe filter unit to sterilize and store at 4 °C.

  6. 6.

    Flt3L/MSA: Sterile MTPBS containing Flt3L at 100 μg/ml and MSA at 1 μg/ml. Store at 4 °C for a maximum of 10 days.

  7. 7.

    B16FLT3L melanoma: Retroviral-mediated gene transfer generated B16-F10 melanoma line secreting murine Flt3L (J. Villadangos, University of Melbourne, Australia) (see Note 2 ).

2.2 Organ Removal

  1. 1.

    FCS: Fetal calf serum. Aliquot and store at −20 °C, or for short periods of time at 4 °C.

  2. 2.

    RPMI-FCS: Modify RPMI-1640 to mouse osmolarity (308 mOsm/kg), and add pH 7.2 HEPES buffering to reduce dependence on CO2 concentration. Adjust to ~pH 7 with CO2, sterilize by running through a 0.2 μM filter unit, and store at 4 °C. Add FCS to a final concentration of 2 % before use.

  3. 3.

    Dissecting instruments (scissors and forceps).

2.3 Digestion of Spleen and Release of DC

  1. 1.

    Enzyme digestion mix: Stock solution (7×) prepared by dissolving collagenase type III (Worthington Biochemicals) at 7 mg/ml and Dnase I (Boehringer Mannheim) at 140 μg/ml in RPMI-FCS. Ensure the collagenase used is free of trypsin and other trypsin-like proteases (see Note 3 ). Divide into 1 ml aliquots and store frozen as a stock solution at −20 °C. Add each 1 ml aliquot to 6 ml RPMI-FCS before use. Run through a 0.2 μM filter unit to sterilize if required. Use immediately.

  2. 2.

    EDTA solution: 0.1 M ethylenediamine tetra-acetic acid disodium salt adjusted to pH 7.2. Run through a 0.2 μM filter unit to sterilize and store at 4 °C.

  3. 3.

    EDTA-FCS: Add 1 ml of 0.1 M EDTA per 10 ml FCS before use.

2.4 Selection of the Light-Density Fraction of Spleen

  1. 1.

    BSS-EDTA: Modified salt solution containing 150 mM NaCl and 3.75 mM KCl (no Ca2+ or Mg2+) and 5 mM EDTA. Adjust to pH 7.2 and mouse osmolarity (308 mOsm/kg). Filter to sterilize using a 0.2 μM filter unit and store at 4 °C.

  2. 2.

    BSS-EDTA-FCS: BSS-EDTA containing 2 % EDTA-FCS.

  3. 3.

    Nycodenz-EDTA: Nycodenz AG powder (Nycomed Pharma AS) is dissolved in water to produce a 0.372 M stock solution and then diluted and adjusted to the desired density of 1.077 g/cm3 at 4 °C and anosmolarity of 308 mOsm/kg using BSS-EDTA (see Note 4 ). Sterilize using a 0.2 μM filter unit and store in 10 ml aliquots at −20 °C. Thaw at room temperature, mix thoroughly, and cool to 4 °C prior to use (see Note 5 ).

  4. 4.

    Polypropylene tubes: 14 ml polypropylene round bottom tubes (Becton Dickinson Labware).

2.5 Depletion of Non-DC Lineages

  1. 1.

    Monoclonal antibody depletion cocktail (see Note 6 ):

    Combine pre-titrated amounts of rat monoclonal antibodies specific for non-DC lineage cells.

    1. (a)

      For purification of cDC only: Add KT3-1.1 (anti-CD3ε), T24/31.7 (anti-CD90), TER119 (anti-erythroid lineage), 1A8 (anti-Ly6G), and RA3-6B2 (anti-CD45R).

    2. (b)

      For purification of both cDC and pDC: Add KT3-1.1 (anti-CD3ε), T24/31.7 (anti-CD90), TER119 (anti-erythroid lineage), 1A8 (anti-Ly6G), and 1D3 (anti-CD19).

    Dilute to the appropriate volume with BSS-EDTA-FCS. Saturating levels of all monoclonal antibodies are used (see Note 7 ). Run through a 0.2 μM filter to sterilize, aliquot, and store at −20 °C.

  2. 2.

    Immunomagnetic beads: BioMag goat anti-rat IgG coated beads (Qiagen).

  3. 3.

    Spiral rotator: Spiramix 10 (Denley).

2.6 In Vitro Administration of Flt3L

  1. 1.

    Red cell lysis medium (RCLM): 0.168 M NH4Cl. Run through a 0.2 μM filter unit to sterilize and store at 4 °C.

  2. 2.

    Flt3L DC culture medium: Modified RPMI-1640, isoosmotic with mouse serum, with additional HEPES buffering at pH 7.2, supplemented with 10 % FCS, 50 μM 2-mercaptoethanol, and 2 mM l-glutamine. Sterilize by running through a 0.2 μM filter unit and store frozen at −70 °C. Add 200 ng/ml murine Flt3L immediately prior to use (see Note 8 ).

  3. 3.

    GM-CSF: Recombinant murine granulocyte macrophage colony-stimulating factor (R&D Systems, Inc.).

2.6.1 Immunofluorescent Staining and Purification via Flow Cytometric Sorting

  1. 1.

    The majority of monoclonal antibodies are purified from hybridoma culture supernatant using Protein G Sepharose (Amersham Biosciences) and subsequently conjugated to fluorochromes in-house. Anti-siglec H (clone eBio440c)-FITC and anti-ESAM (clone 1G8)-PE conjugates are purchased from eBioscience. They are all titrated to determine saturating levels.

  2. 2.

    mAbs are conjugated to fluorochromes and biotin following the manufacturer’s instructions:

    1. (a)

      Conjugate P84 (anti-CD172a), 14.8 (anti-CD45RA), RA36B2 (anti-CD45R), and 120G8 (anti-CD317) to FITC (Molecular Probes, Inc.).

    2. (b)

      M2/90 (anti-CD103) to phycoerythrin (PE) (ProZyme).

    3. (c)

      M1/70 (anti-CD11b) to AlexaFluor680 (Molecular Probes, Inc.).

    4. (d)

      N418 (anti-CD11c) and YTS169.4 (anti-CD8) to allophycocyanin (APC) (ProZyme).

    5. (e)

      M1/69 (anti-CD24) to Pacific Blue (Molecular Probes, Inc.).

    6. (f)

      N418 (anti-CD11c) to PerCp.Cy5.5 (Innova Biosciences).

    7. (g)

      GK1.5 (anti-CD4), 10B4 (anti-Clec9A), and 5D3 (anti-Clec12A) to biotin (Molecular Probes, Inc.).

    Add FCS to 1 % and NaN3 to a final concentration of 10 mM (see Note 9 ). Titrate to determine saturating levels. Aliquot stocks of FITC, biotin, AlexaFluor680, and Pacific Blue conjugates, and store at −70 °C. Stocks of PE, APC, and PerCp.Cy5.5 conjugates (see Note 10 ) and working stocks of FITC, biotin, AlexaFluor680, and Pacific Blue conjugates are stored at 4 °C, protected from light. Dilute to their final working concentration immediately prior to use.

  3. 3.

    PI: 100 μg/ml propidium iodide (Calbiochem) stock solution in normal saline. Aliquot and store at 4 °C protected from light (see Note 11 ).

  4. 4.

    BSS-EDTA-FCS-PI: Working solution of PI made by diluting the PI stock in BSS-EDTA-FCS to a final working concentration of 500 ng/ml before addition to cells.

  5. 5.

    FACSAria (BD Biosciences).

2.6.2 Immunofluorescent Staining and Purification via Immunomagnetic Beads

  1. 1.

    Fluorochrome-conjugated mAb:

    1. (a)

      Conjugate YTS169.4 and M1/69 to FITC (Molecular Probes, Inc.).

    2. (b)

      Conjugate M1/70 to phycoerythrin (PE) (ProZyme).

  2. 2.

    Anti-fluorochrome beads (Miltenyi Biotec):

    1. (a)

      Anti-FITC microbeads.

    2. (b)

      Anti-PE microbeads.

  3. 3.

    BSS-EDTA-0.5 %FCS: BSS-EDTA containing 0.5 % EDTA-FCS.

  4. 4.

    MACS column (Miltenyi Biotec):

    1. (a)

      MACS LS column (×2).

    2. (b)

      MACS LD column.

  5. 5.

    Magnet and stand (Miltenyi Biotec):

    1. (a)

      Mini MACS magnet.

    2. (b)

      MACS multistand.

2.6.3 Staining and Purification via Immunomagnetic Beads: Removal of NK Cells

  1. 1.

    Conjugated mAb: Conjugate DX5 to biotin (Molecular Probes, Inc.).

  2. 2.

    Immunomagnetic beads (Miltenyi Biotec): anti-biotin microbeads.

  3. 3.

    BSS-EDTA-0.5 %FCS: BSS-EDTA containing 0.5 % EDTA-FCS.

  4. 4.

    MACS column (Miltenyi Biotec): MACS LD column.

  5. 5.

    Magnet and stand (Miltenyi Biotec):

    1. (a)

      Mini MACS magnet.

    2. (b)

      MACS multistand.

3 Methods

3.1 In Vivo Administration of Flt3L

3.1.1 Soluble Flt3L

  1. 1.

    Inject each mouse daily for 10 days with 100 μl of Flt3L/MSA subcutaneously into the nape of the neck using a 27G needle (see Note 12 ).

3.1.2 B16Flt3L

  1. 1.

    Maintain the B16Flt3L melanoma in in vitro culture in RPMI-FCS.

  2. 2.

    Pour off the supernatant from the culture flasks, and wash with 5 ml of MTPBS.

  3. 3.

    Pour off the MTPBS, add 2 ml of trypsin, and treat at 37 °C for 2 min.

  4. 4.

    Shake the flask to dislodge adherent cells and remove and pool cells from multiple flasks.

  5. 5.

    Centrifuge, resuspend the pellet in MTPBS, and count.

  6. 6.

    Dilute cells at 25 × 106/ml in MTPBS.

  7. 7.

    Inject 200 μl (5 × 106) cells per mouse subcutaneously into the nape of the neck using a 27G needle (see Note 13 ).

  8. 8.

    Monitor injected mice daily (see Note 14 ).

  9. 9.

    Allow melanoma to grow for up to 10 days (see Note 15 ).

3.2 Organ Removal

  1. 1.

    Remove spleens from eight untreated mice or from two Flt-3L-treated mice (see Note 16 ) into cold RPMI-FCS, taking care to remove the organs with as little fat and connective tissue as possible.

3.3 Digestion of Spleen and Release of DC

  1. 1.

    Prepare the enzyme digestion mix slightly ahead of time, and allow it to warm to room temperature before use.

  2. 2.

    Remove any remaining fat and/or connective tissue (see Note 17 ) from the spleens using two 20G needles, and transfer spleens to a small Petri dish containing 7 ml of enzyme digestion mix. Use a sharp pair of scissors or a single-sided razor blade to cut the tissue into very small fragments (see Note 18 ). Transfer the fragments to a 10 ml tube using a wide-bore Pasteur pipette. Mix frequently, using the same pipette, while digesting the tissue for 20–25 min at room temperature (~22 °C) (see Note 19 ).

  3. 3.

    Add 600 μl of EDTA solution to the digestion mix, and continue the incubation for a further 5 min (see Note 20 ).

  4. 4.

    Run the digestion mix through a sieve to remove any remaining undigested tissue. Discard anything caught in the sieve. Dilute the digestion mix to 9 ml with RPMI-FCS, underlay with 1 ml of cold FCS-EDTA, and centrifuge to recover the cells (see Note 21 ).

3.4 Selection of the Light-Density Fraction of Spleen

  1. 1.

    Thaw two 10 ml aliquots of Nycodenz-EDTA at room temperature. Once thawed, mix thoroughly and keep at 4 °C until required (see Note 22 ).

  2. 2.

    Resuspend the cell pellet in 10 ml of Nycodenz-EDTA (see Note 23 ).

  3. 3.

    Transfer 5 ml of the remaining Nycodenz-EDTA into the bottom of each of two polypropylene tubes.

  4. 4.

    Gently layer 5 ml of the cell suspension over the Nycodenz-EDTA in each of the two tubes (see Note 24 ). Add a 1–2 ml layer of EDTA-FCS over the cell suspension.

  5. 5.

    Disrupt the interface gently by inserting the tip of a Pasteur pipette, swirling and removing it (see Note 25 ).

  6. 6.

    Perform the density cut in a swing-out head, refrigerated centrifuge, set at 4 °C, for 10 min at 1700 × g with the brake set on low.

  7. 7.

    Collect the light-density fraction in the upper zones down to the 4 ml mark using a Pasteur pipette (see Note 26 ). Discard the bottom 4 ml and the cell pellet.

  8. 8.

    Transfer the light-density fraction to a 50 ml tube and dilute up to 50 ml with BSS-EDTA. Mix thoroughly and centrifuge to wash and recover the cells (see Note 27 ).

  9. 9.

    Resuspend the cells in 5 ml BSS-EDTA-FCS and count (see Note 28 ). Calculate the total cell number recovered.

3.5 Depletion of Non-DC Lineages

  1. 1.

    Calculate the volume of monoclonal antibody depletion cocktail required if 10 μl is needed per 106 cells.

  2. 2.

    Add the required volume of the appropriate monoclonal antibody depletion cocktail (see Note 29 ) to the cell pellet and resuspend and incubate at 4 °C for 30 min.

  3. 3.

    Calculate the required volume of immunomagnetic beads required (see Note 30 ) and transfer them to 5 ml polypropylene tubes. Wash the beads by diluting with BSS-EDTA-FCS (see Note 31 ), placing the tubes into the magnet, allowing the beads to move to the magnet and removing the supernatant. Repeat the washing step three to four times. After the final wash, pellet the beads at the bottom of the tube in a small amount of BSS-EDTA-FCS and place the tube at 4 °C until required.

  4. 4.

    Dilute the cells up to 9 ml with BSS-EDTA-FCS and underlay with 1 ml of FCS-EDTA. Centrifuge the cells and remove the supernatant from the top, leaving the FCS layer over the cells (see Note 32 ). Pulse in the centrifuge for 15 s to force any remaining supernatant down the wall of the tube. Then remove any supernatant and the FCS (see Note 33 ). Resuspend the cells in 400–500 μl of BSS-EDTA-FCS.

  5. 5.

    Remove the BSS-EDTA-FCS from the pellet of immunomagnetic beads and add the cells. Vortex the tube very briefly to produce a bead-cell slurry (see Note 34 ). Seal the tube and mix the slurry continuously for 20 min at 4 °C at an angle of 30° on a spiral mixer (see Note 35 ).

  6. 6.

    Dilute the slurry with 3 ml of BSS-EDTA-FCS, mix very gently, and attach the tube to the magnet for 2 min.

  7. 7.

    Recover the supernatant containing unbound DC with a Pasteur pipette, and transfer to a second 5 ml polypropylene tube. Discard the tube containing magnetic beads bound to non-DC (see Note 36 ). Place the tube containing the supernatant into the magnet for a further 2 min to remove any remaining beads. Transfer the supernatant to a 10 ml tube.

  8. 8.

    Layer 1 ml of FCS-EDTA under the cell suspension and centrifuge to recover the DC fraction. Resuspend the cells in 2 ml of BSS-EDTA-FCS and count.

  9. 9.

    Maintain the cells at 4 °C until they are required for immunofluorescent labeling.

3.6 In Vitro Administration of Flt3L

  1. 1.

    Remove the femurs and tibiae from the desired number of mice, removing as much tissue and sinew as possible, and place in RPMI-FCS at 4 °C (see Note 37 ).

  2. 2.

    Use scissors to remove the top of each bone to allow access to the bone marrow. Control each bone with a pair of sterile forceps, and flush the bone with RPMI-FCS using a 23G needle and syringe to remove the bone marrow. Pool the bone marrow from individual bones. Mix the suspension up and down in a syringe and 26G needle to create a single cell suspension. Underlay with FCS and centrifuge.

  3. 3.

    Remove the supernatant and gently resuspend the cells in 500 μl of RCLM per mouse used. Expose the cells to RCLM for 30 s (see Note 38 ). Dilute the cells immediately with RPMI-FCS, pass them into a new tube through a sieve to remove clumps, underlay with FCS, and wash by centrifugation. Repeat the washing step twice more. Resuspend the cells and count.

  4. 4.

    Centrifuge the cells and resuspend the pellet at 1.5 × 106/ml in Flt3L culture medium. If desired add 300pg/ml GM-CSF to boost numbers at days 8 and 9 of culture (see Note 39 ). Culture the cells for 8–9 days at 37 °C in 10 % CO2 in air (see Note 40 ).

  5. 5.

    If required, add 5 ng/ml GM-CSF at day 6 of culture, to increase the proportion of CD103+ cDC at day 8 or 9.

  6. 6.

    At the completion of the culture period, harvest the cells by gently washing the flasks several times with cold BSS-EDTA-FCS (see Note 41 ). Centrifuge to recover the cells and resuspend the pellet in BSS-EDTA-FCS. Count to determine recovery.

  7. 7.

    Keep the cell suspension at 4 °C until the cells are required for immunofluorescent labeling.

3.7 Immuno-fluorescent Staining and Purification

3.7.1 Via Flow Cytometric Sorting

  1. 1.

    Prepare a cocktail of pre-titrated fluorochrome-conjugated monoclonal antibodies at the appropriate concentration immediately prior to use.

  2. 2.

    Centrifuge to recover the cells and remove the supernatant.

  3. 3.

    Add the fluorochrome-conjugated antibody cocktail at 10 μl per 106 cells, resuspend by flicking the tube, and incubate at 4 °C for 30 min (see Note 42 ).

  4. 4.

    Resuspend up to a larger volume with BSS-EDTA-FCS and underlay with FCS-EDTA. Centrifuge to wash the cells.

  5. 5.

    Remove the supernatant leaving the FCS-EDTA layer above the cells. Pulse the tube in the centrifuge for 15 s to force any remaining media to wash down the wall of the tube. Remove the remaining media and the FCS-EDTA layer.

  6. 6.

    Resuspend the cells in BSS-EDTA-FCS-PI and keep cells at 4 °C until they are required for flow cytometry (see Note 43 ).

  7. 7.

    Immediately prior to sorting, add the cell suspension to a syringe fitted with a 26G needle. Gently force the cells through the needle and into a cell strainer cap fitted on a 5 ml polystyrene round bottom tube. Allow the cells to run through the strainer and collect in the tube.

  8. 8.

    Sort the DC using a FACSAria (see Note 44 ). The flow cytometer should be set up with standard lasers in place: a blue 488 nm emitting laser for the detection of FITC, PE, PI, PerCp.Cy5.5, and PE.Cy7; a violet/near UV laser, emitting wavelengths of 375 and 405 nm, to detect Pacific Blue; and a red laser emitting a wavelength of 640 nm to detect APC, as well as the appropriate filters and dichroic mirrors. Select DC on the basis of high forward and side light scatter, excluding dead cells with high PI fluorescence . Contaminating macrophages should be removed by gating out autofluorescent cells using the PI channel in combination with another unused fluorescence channel (see Note 45 ). Identify CD11cint siglec-H+ pDC and CD11chisiglec-H− cDC (see Note 46 ). Use combinations of conjugated antibodies to identify and sort other dendritic cell subtypes (see Note 47 ).

3.7.2 Via Immunomagnetic Beads

  1. 1.

    Centrifuge cells to a pellet.

  2. 2.

    Add YTS169.4-FITC (anti-CD8) (see Note 48 ) at 10 μl/106 cells (see Note 49 ) and resuspend and incubate at 4 °C for 20 min (see Note 50 ).

  3. 3.

    Dilute up to a larger volume with BSS-EDTA-FCS, underlay with FCS-EDTA, and centrifuge to recover the cells.

  4. 4.

    Remove the supernatant leaving the FCS-EDTA layer above the cells and pulse in the centrifuge for 15 s to force any remaining media down the wall of the tube. Remove the remaining media and the FCS-EDTA layer.

  5. 5.

    Prepare anti-FITC microbeads (see Note 51 ) at 1 μl/4 × 106 cells in a final volume of 2.5 μl/106 cells BSS-EDTA-0.5 %FCS (see Note 52 ), and add to the cell pellet. Resuspend gently by flicking the tube.

  6. 6.

    Incubate for 15 min in a 4 °C cold room (see Note 53 ).

  7. 7.

    Dilute to a larger volume with BSS-EDTA-FCS and underlay with FCS-EDTA. Centrifuge the cells to wash away unbound microbeads.

  8. 8.

    Equilibrate a MACS LS column (see Note 54 ) by placing it into a cold miniMACS magnet suspended on a MACS multistand and washing with 3 ml BSS-EDTA-0.5 %FCS (see Note 55 ).

  9. 9.

    Resuspend the cells in 3 ml BSS-EDTA-0.5 %FCS and apply them to the column. Rinse the column with 3 ml of BSS-EDTA-0.5 %FCS. Add 5 ml of BSS-EDTA-0.5 %FCS to the column and collect the flow through. Run another 5 ml of BSS-EDTA-0.5 %FCS through the column, and pool the flow through containing the unbound CD8− cells.

  10. 10.

    Remove the column from the magnet, add 5 ml BSS-EDTA-0.5 %FCS, and allow to run to wash the previously bound CD8+ cells from the column. Add a further 5 ml of BSS-EDTA-0.5 % FCS to the column. Use the supplied plunger to force all liquid from the column to ensure all cells are removed. Pool, count, and set aside the recovered CD8+ cells at 4 °C. Discard the column.

  11. 11.

    Count the CD8− cells and centrifuge them.

  12. 12.

    Add M1/69-FITC (anti-CD24) at 10 μl/106 cells and resuspend and incubate at 4 °C for 20 min (see Note 56 ).

  13. 13.

    Dilute to a larger volume with BSS-EDTA-FCS and underlay with FCS-EDTA. Centrifuge the cells.

  14. 14.

    Remove the supernatant leaving the FCS-EDTA layer above the cells, and pulse in the centrifuge for 15 s to force any remaining media to wash down the wall of the tube. Remove the remaining media and the FCS-EDTA layer.

  15. 15.

    Prepare anti-FITC microbeads at 1 μl/4 × 106 cells in a final volume of 2.5 μl/106 cells BSS-EDTA-0.5 %FCS and add to the cell pellet. Resuspend gently by flicking the tube.

  16. 16.

    Repeat steps 6 and 7.

  17. 17.

    Equilibrate (see Note 54 ) a MACS LD column (see Note 57 ) by placing into a cold miniMACS magnet suspended on a MACS multistand and washing with 2 ml BSS-EDTA-0.5 %FCS (see Note 58 ).

  18. 18.

    Resuspend the cells in 1 ml BSS-EDTA-0.5 %FCS and apply them to the column. Rinse the column with 1 ml of BSS-EDTA-0.5 %FCS. Repeat the wash once more, and collect the flow through containing the unbound CD24− CD8− fraction of cells. Discard the column (see Note 59 ).

  19. 19.

    Count the CD24−CD8− cells and centrifuge them.

  20. 20.

    Add M1/70-PE (anti-CD11b) at 10 μl/106 cells and resuspend and incubate at 4 °C for 20 min.

  21. 21.

    Dilute up to a larger volume with BSS-EDTA-FCS and underlay with FCS-EDTA. Centrifuge to recover the cells.

  22. 22.

    Remove the supernatant leaving the FCS-EDTA layer above the cells and pulse in the centrifuge for 15 s to force any remaining media to wash down the wall of the tube. Remove the remaining media and the FCS-EDTA layer.

  23. 23.

    Prepare anti-PE microbeads at 1 μl/4 × 106 cells in a final volume of 2.5 μl/106 cells BSS-EDTA-0.5 %FCS and add to the cells. Gently resuspend by flicking the tube.

  24. 24.

    Repeat steps 6–8.

  25. 25.

    Resuspend the cells in 3 ml BSS-EDTA-0.5 %FCS and apply them to the column. Rinse the column with 3 ml of BSS-EDTA-0.5 %FCS. Add 5 ml of BSS-EDTA-0.5 %FCS to the column and collect the flow through. Run another 5 ml of BSS-EDTA-0.5 %FCS through the column and discard the flow through containing the unbound CD24− CD8− CD11b− fraction of cells.

  26. 26.

    Remove the column from the magnet and add 5 ml BSS-EDTA-0.5 %FCS to wash the previously bound CD24− CD8− CD11b+ cells from the column. Add a further 5 ml of BSS-EDTA-0.5 %FCS. Use the supplied plunger to force all liquid from the column to ensure all cells are removed. Count the recovered CD24− CD8− CD11b+ cells (see Note 60 ).

3.7.3 Depletion of NK Cells via Immunomagnetic Beads

  1. 1.

    If depletion of NK cells is deemed necessary, centrifuge cells to a pellet.

  2. 2.

    Add saturating levels of DX5-biotin at 10 μl/106 cells, and resuspend and incubate at 4 °C for 30 min. Dilute up to a larger volume with BSS-EDTA-FCS, underlay with FCS-EDTA, and centrifuge to recover the cells.

  3. 3.

    Remove the supernatant leaving the FCS-EDTA layer above the cells, and pulse in the centrifuge for 15 s to force any remaining media down the wall of the tube. Remove the remaining media and the FCS-EDTA layer.

  4. 4.

    Prepare anti-biotin microbeads (see Note 51 ) at 1 μl/4 × 106 cells in a final volume of 2.5 μl/106 cells BSS-EDTA-0.5 %FCS (see Note 52 ), and add to the cell pellet. Resuspend gently by flicking the tube.

  5. 5.

    Incubate for 15 min in a 4 °C cold room (see Note 53 ).

  6. 6.

    Dilute to a larger volume with BSS-EDTA-FCS and underlay with FCS-EDTA. Centrifuge the cells to wash away unbound microbeads.

  7. 7.

    Equilibrate a MACS LD column (see Notes 54 and 58 ) by placing it into a cold miniMACS magnet suspended on a MACS multistand and washing with 3 ml BSS-EDTA-0.5 %FCS (see Note 55 ).

  8. 8.

    Resuspend the cells in 1 ml BSS-EDTA-0.5 %FCS and apply them to the column. Rinse the column with 1 ml of BSS-EDTA-0.5 %FCS. Repeat the wash once more and collect the flow through containing the unbound DX5− DC fraction of cells. Discard the column.

4 Notes

  1. 1.

    Alternatively use recombinant human Flt3L.

  2. 2.

    The B16Flt3L melanoma was derived from male C57/BL6 mice. Injection into mice of other strains, or into female mice, may elicit an immune response and affect the DC recovered.

  3. 3.

    The level of contamination of collagenase with trypsin or trypsin-like proteases can vary between batches, so each should be tested prior to use. Proteases can strip cell surface molecules and alter the surface phenotype of the DC. We test each new batch of collagenase for the presence of these proteases by using them to digest thymocytes for 30 min at 37 °C and then screening for the loss of the trypsin-sensitive cell surface markers CD4 and CD8 by flow cytometry .

  4. 4.

    A pycnometer is used to determine the density of the Nycodenz accurately, by reference to water, using an analytical balance. The pycnometer is a glass flask with a close-fitting ground-glass lid with a capillary tube in it, which allows air bubbles or excess Nycodenz to escape from the vessel. The pycnometer is weighed empty, full of water, and full of Nycodenz and the specific gravity of the Nycodenz calculated. A correction needs to be made as the density of water will not be 1 g/cm3 at 4 °C.

  5. 5.

    Temperature, pH, and osmolarity all affect the buoyant density of cells, so Nycodenz of higher, or lower, than recommended density will affect the purity and the yield of recovered cells. We calculate the density of Nycodenz at pH 7.2, 308 mOsm/kg, and 4 °C. Temperature is of particular importance during the density cut, so care must be taken to ensure the Nycodenz and the centrifuge to be used for the density cut are at 4 °C.

  6. 6.

    The monoclonal antibodies we have included in the cocktail for depletion of non-DC lineage cells and those we have utilized to identify DC and DC subpopulations are available commercially.

  7. 7.

    Appropriate levels should be determined for each batch of antibody to be included in the cocktail. The rat anti-mouse monoclonal antibodies are individually titrated via flow cytometry using a fluorochrome-conjugated anti-rat Ig secondary reagent in order to determine their working dilution. Antibodies are added to ensure surface saturating quantities of each are contained in 10uL of cocktail. Concentrations determined to result in cell surface saturation of the antigen are considered adequate for efficient depletion and are used in the cocktail.

  8. 8.

    Flt3L should be titrated prior to use by small-scale bone marrow culture. A range of 50–300 ng/ml should be tested. Suboptimal levels will vastly reduce DC yield. Optimal levels can result in DC recoveries of up to 130 % of the starting number of bone marrow cells.

  9. 9.

    All proper precautions should be taken when using sodium azide, particularly when preparing the stock solution. Adequate protective clothing, including safety glasses, gloves, and face mask, should be worn. Sodium azide is extremely toxic if ingested.

  10. 10.

    Do not freeze phycoerythrin (PE), allophycocyanin (APC), and PerCp.Cy5.5 or their conjugates. They are extremely sensitive to freezing and thawing and will lose activity.

  11. 11.

    All proper precautions including protective clothing, safety glasses, gloves, and face mask should be worn during preparation of the stock solution of propidium iodide. Propidium iodide is an irritant and potentially toxic.

  12. 12.

    MSA is used as a carrier protein to minimize loss of Flt3L sticking to tubes, syringes, etc. It may be possible to substitute with endotoxin-free BSA (bovine serum albumin) or even FCS (fetal calf serum), but care must be taken not to elicit an immune response against the carrier protein.

  13. 13.

    Aim to insert the needle and inject as centrally between the shoulder blades as possible. The melanoma will develop rapidly into a large growth and if not located centrally will make normal movement very difficult for the mouse.

  14. 14.

    Mice should be monitored regularly and any showing signs of distress or illness euthanized.

  15. 15.

    The rate of growth of the melanoma should not adversely affect the mice for at least 10 days after injection. After 10 days, however, mice will begin to show signs of distress and/or illness. It is therefore recommended that the melanoma should not be allowed to develop for longer than 10 days.

  16. 16.

    Provision has been made to cater for the greatly increased size of spleens treated with Flt3L. A proportional increase or decrease of all listed amounts and volumes should also be made to cater for any change in the starting number of organs.

  17. 17.

    Residual fat will reduce cell viability and, combined with undigested connective tissue, will accumulate and cause clumping. It is therefore important to clean the organs as much as possible before commencement of the digestion. We use two 20G needles to perform the cleaning, but any suitable instrument may be used.

  18. 18.

    The organs are cut into very small fragments to increase the surface area available to the enzymes. This ensures adequate digestion and maximizes cell yield.

  19. 19.

    Inadequate digestion will result in a lower recovery and the preferential loss of certain DC populations that are more firmly entrenched in the tissue. A digestion time of 20 min should prove sufficient to digest all but the pulpy tissue from spleen, provided the tissue was cut up adequately prior to the digestion and adequate mixing occurred during the digestion. The digestion may be extended to 25 min if required.

  20. 20.

    EDTA inhibits collagenase and effectively ends the digestion. EDTA also chelates Ca2+ and Mg2+ ions and will dissociate lymphocyte-DC complexes. EDTA must be added to all media from this point onward to stop the reformation of these multicellular complexes. Failure to do so will cause loss of DC during purification and possible contamination of the recovered DC with lymphocytes.

  21. 21.

    All centrifugation steps are performed at 1000 × g for 7 min at 4 °C unless otherwise stated. Underlaying the sample with FCS, thereby incorporating a zonal centrifugation step, increases the efficiency of separation of cells from smaller particles and soluble material in the supernatant. It therefore eliminates the need for repetitive “washing” of the cells.

  22. 22.

    Nycodenz has a tendency to settle over time, so mix it thoroughly prior to aliquoting and again prior to use to ensure a solution of uniform density.

  23. 23.

    Efficiency of separation will be lost and yields reduced if the density separation is overloaded. Do not load more than four untreated or one Flt3L treated spleen (5 × 108–109 cells) per 10 ml of Nycodenz.

  24. 24.

    A discrepancy between the density of the Nycodenz at the top of an aliquot and the bottom, or between different aliquots (most likely due to inadequate mixing), will affect the ability to layer Nycodenz containing cells over the Nycodenz at the bottom of the tube. Ensure Nycodenz has been adequately mixed and is of uniform density before use.

  25. 25.

    Disruption of the interface creates a density gradient rather than a sharp band and increases the efficiency of the density separation.

  26. 26.

    The light-density fraction of cells will be found as a band at the interface zone between the FCS and Nycodenz, while dense cells will have formed a pellet. Cells of intermediate density will be found in the gradient between these zones, so collect all cells down to the 4 ml mark while concentrating on the light-density band at the interface. The 4 ml mark is an arbitrary one, and collecting a few ml either side of this will not alter recovery greatly. It is just important to be consistent.

  27. 27.

    Adequate dilution and mixing of the light-density fraction of cells with EDTA-BSS is essential to recover them as a pellet during centrifugation.

  28. 28.

    This count is used to calculate the appropriate volume of mAb depletion cocktail and immunomagnetic beads required in subsequent steps. It is important that all cells, including any remaining erythrocytes, are included in the count. As a rough guide, the light-density fraction should represent 5–7 % of the starting number of cells.

  29. 29.

    Anti-CD45R is included in the cocktail to deplete B cells but will also deplete pDC, which are all CD45R+. In order to include pDC, but continue to deplete B cells, replace anti-CD45R with anti-CD19.

  30. 30.

    For the isolation of spleen DC, we recommend BioMag beads at a 10:1 bead-to-cell ratio. BioMag beads provide optimal economy and reasonably good efficiency.

  31. 31.

    Immunomagnetic beads must be washed prior to use to remove preservative.

  32. 32.

    Centrifuging through a layer of FCS separates cells from unbound (excess) mAb.

  33. 33.

    It is important to carefully remove all the supernatant after washing as any remaining unbound mAb will compete for binding to the beads thus decreasing the efficiency of the depletion.

  34. 34.

    The efficiency of the depletion is greatly increased by maximizing contact between beads and cells in the concentrated slurry.

  35. 35.

    Fitting a wide ring around the top of the tube increases the angle of rotation to 30° from the horizontal and keeps the bead-cell slurry concentrated at the bottom of the tube. The wide ring also serves to slow the rate of rotation.

  36. 36.

    If numbers are critical, more DC can be recovered by washing the beads and attaching the tube to the magnet a second time; however, this will result in reduced purity.

  37. 37.

    We routinely harvest 4–7 × 107 cells per mouse.

  38. 38.

    RCLM is toxic so it should be added and mixed gently and then washed away quickly. An exposure of 15–30 s is sufficient to lyse erythrocytes in bone marrow.

  39. 39.

    The optional addition of 300 pg/ml GM-CSF to the Flt3L culture medium will improve DC yield.

  40. 40.

    Maximum yield of the DC subpopulations can also vary with culture time, so an optimal culture time should be determined for each batch of Flt3L. Production of pDC peaks at day 8 and cDC production at day 9 of culture.

  41. 41.

    This gentle washing step should be sufficient to remove any slightly adherent DC from the plastic but leave behind any more adherent macrophages .

  42. 42.

    All immunostaining steps should be performed at 4 °C to promote cell viability and to prevent capping of monoclonal antibody from the cell surface.

  43. 43.

    Propidium iodide is used for dead cell exclusion during flow cytometric analysis.

  44. 44.

    Any flow cytometer with sorting capabilities and appropriate lasers and optical setup can be used.

  45. 45.

    Remove autofluorescent cells (mainly macrophages ) during fluorescence activated cell sorting or analysis by gating out cells that have low levels of fluorescence in two or more fluorescent channels. Ideally use a combination of PI and an unused channel. During multicolor sorting or analysis, it may be necessary to combine autofluorescence in the PI channel with low fluorescence in a channel that is being used. Choose a parameter where all DC will fluoresce brightly (i.e., CD11c) and gate out cells of low fluorescence .

  46. 46.

    CD45RA, CD45R (B220), and CD317 are alternative markers to siglec H and are often used to separate pDC from cDC.

  47. 47.

    If functional studies are to be undertaken, the cells should be washed to remove propidium iodide post sorting.

  48. 48.

    After Flt-3L treatment, it is advisable to purify the more abundant CD8+ fraction first in order to avoid contamination of the CD11b+ fraction.

  49. 49.

    Too high a concentration of antibody added at this stage tends to increase the nonspecific loss of DC and lower the yield. We routinely use antibodies at a quarter to a half their saturating levels, but would recommend that each user carefully titrate their antibody for optimal performance.

  50. 50.

    It is not important what fluorochrome the anti-CD8 monoclonal antibody is conjugated to as long as an appropriate anti-fluorochrome bead is available.

  51. 51.

    Although directly coupled anti-CD8 beads are available, we find that efficiency of separation is lowered when they are used.

  52. 52.

    This is a lower bead-to-cell ratio than recommended by the manufacturer. We have compensated for this by reducing the volume in order to increase the final concentration and maximize bead-to-cell contact. This has resulted in a more economical and efficient process.

  53. 53.

    Incubation may also be completed in a refrigerator; however, ice should be avoided as temperatures below 4 °C will decrease binding .

  54. 54.

    Do not overload the column. This will result in a lower recovery and reduced purity. An LS column can accommodate up to 5 × 108 cells with a maximum of 108 positive cells and an LD column up to 109 cells with a maximum of 5 × 108 positive cells. Ensure accurate counts of cells are made and appropriate numbers of columns used.

  55. 55.

    Cell viability is increased at 4 °C, so cooling the magnet before use ensures that the column and cell suspension passing through it are kept cold during the separation. Placing the magnet at −20 °C and the columns at 4 °C for 15 min before use is recommended.

  56. 56.

    A second depletion for CD8-bearing cells using anti-CD24 conjugated to FITC is necessary to improve purity of the CD11b+ cells. This depletion also removes the CD8−CD24+ precursors of the CD8+ DC lineage.

  57. 57.

    LS columns are designed for selection and LD columns for depletion. So when positively selecting a population (CD8+ or CD11b+), we use an LS column, and when trying to select a population we wish to discard (CD24+), we use an LD column.

  58. 58.

    LD columns are more tightly packed and will run much more slowly than LS columns. Allow sufficient time for equilibration of the column, and be aware that the longer exposure of cells to room temperature while they are running through the column may affect purity and/or viability. Consider running LD columns in a cold room.

  59. 59.

    As the bound fraction of cells in this case will be a mix of CD8+CD24+ and CD8−CD24+ cells, it is typically discarded. If this fraction is required, it may be recovered using the typical washing procedure used to recover the CD8+ and CD11b+ fractions.

  60. 60.

    Purity of all collected populations may be tested. Fluorochromes on the surface of the cells are not affected by bound beads and can be readily detected using flow cytometry .