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Cytotechnology

, Volume 70, Issue 6, pp 1685–1695 | Cite as

Development of pipette tip gap closure migration assay (s-ARU method) for studying semi-adherent cell lines

  • Swapnil Ganesh Sanmukh
  • Sérgio Luis FelisbinoEmail author
Method in Cell Science

Abstract

This work presents a pipette tip gap closure migration assay prototype tool (semi-adherent relative upsurge—s-ARU—method) to study cell migration or wound healing in semi-adherent cell lines, such as lymph node carcinoma of the prostate (LNCaP). Basically, it consists of a 6-well cover plate modification, where pipette tips with the filter are shortened and fixed vertically to the inner surface of the cover plate, with their heights adjusted to touch the bottom of the well center. This provides a barrier for the inoculated cells to grow on, creating a cell-free gap. Such a uniform gap formed can be used to study migration assay for both adherent as well as semi-adherent cells. After performing time studies, effective measurement of gap area can be carried out conveniently through image analysis software. Here, the prototype was tested for LNCaP cells, treated with testosterone and flutamide as well as with bacteriophages T4 and M13. A scratch assay using PC3 adherent cells was also performed for comparison. It was observed that s-ARU method is suitable for studying LNCaP cells migration assay, as observed from our results with testosterone, flutamide, and bacteriophages (T4 and M13). Our method is a low-cost handmade prototype, which can be an alternative to the other migration assay protocol(s) for both adherent and semi-adherent cell cultures in oncological research along with other biological research applications.

Keywords

LNCaP Migration assay Semi-adherent cell Wound healing Gap closure assay 

Abbreviations

s-ARU

Semi-adherent relative upsurge

PC3

Prostate cancer cell line

LNCaP

Lymph node carcinoma of the prostate

ECM

Extracellular matrix

ATCC

American Type Culture Collection

Notes

Acknowledgements

This article comprises part of the Ph.D. thesis of Swapnil Ganesh Sanmukh, supported by a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Programa de Estudantes-Convênio de Pós-Graduação (PEC-PG) funding (Process Number 8963-14-2 PEC-PG 2014). SLF received a grant from the Sao Paulo Research Foundation (FAPESP) Ref. 2016/09532-3 and from CNPq Ref. 305391/2014-3. SGS would like to dedicate this work to his daughter Miss. Arundhati (ARU) Sanmukh.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

References

  1. Albini A (1998) Tumor and endothelial cell invasion of basement membranes. The matrigel chemoinvasion assay as a tool for dissecting molecular mechanisms. Pathol Oncol Res 4:230–241CrossRefGoogle Scholar
  2. Allen WE, Zicha D, Ridley AJ, Jones GE (1998) A role for Cdc42 in macrophage chemotaxis. J Cell Biol 141:1147–1157CrossRefGoogle Scholar
  3. Anand-Apte B, Zetter BR, Viswanathan A, Qiu RG, Chen J, Ruggieri R, Symons M (1997) Platelet-derived growth factor and fibronectin-stimulated migration are differentially regulated by the Rac and extracellular signal-regulated kinase pathways. J Biol Chem 5:30688–30692CrossRefGoogle Scholar
  4. Banyard J, Anand-Apte B, Symons M, Zetter BR (2000) Motility and invasion are differentially modulated by Rho family GTPases. Oncogene 19:580–591CrossRefGoogle Scholar
  5. Belo VA, Guimarães DA, Castro MM (2016) Matrix metalloproteinase 2 as a potential mediator of vascular smooth muscle cell migration and chronic vascular remodeling in hypertension. J Vasc Res 52:221–231CrossRefGoogle Scholar
  6. Burridge K, Wennerberg K (2004) Rho and Rac take center stage. Cell 116:167–179CrossRefGoogle Scholar
  7. Cai G, Lian J, Shapiro SS, Beacham DA (2000) Evaluation of endothelial cell migration with a novel in vitro assay system. Methods Cell Sci 22:107–114CrossRefGoogle Scholar
  8. Dabrowska K, Skaradziński G, Jończyk P, Kurzepa A, Wietrzyk J, Owczarek B, Zaczek M, Switała-Jeleń K, Boratyński J, Poźniak G, Maciejewska M, Górski A (2009) The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration. BMC Microbiol 9:13CrossRefGoogle Scholar
  9. DeVries ME, Ran L, Kelvin DJ (1999) On the edge: the physiological and pathophysiological role of chemokines during inflammatory and immunological responses. Semin Immunol 11:95–104CrossRefGoogle Scholar
  10. Fidler IJ (2002) Critical determinants of metastasis. Semin Cancer Biol 12:89–96CrossRefGoogle Scholar
  11. Geback T, Schulz MM, Koumoutsakos P, Detmar M (2009) TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays. Biotechniques 46:265–274CrossRefGoogle Scholar
  12. Gupta GP, Massague J (2006) Cancer metastasis: building a framework. Cell 127:679–695CrossRefGoogle Scholar
  13. Heldin CH, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316CrossRefGoogle Scholar
  14. Hood JD, Cheresh DA (2002) Role of integrins in cell invasion and migration. Nat Rev Cancer 2:91–100CrossRefGoogle Scholar
  15. Hulkower KI, Herber RL (2011) Cell migration and invasion assays as tools for drug discovery. Pharmaceutics 3:107–124CrossRefGoogle Scholar
  16. Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T, Narumiya S (1999) An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med 5:221–225CrossRefGoogle Scholar
  17. Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV (2014) In vitro cell migration and invasion assays. J Vis Exp 88:e51046Google Scholar
  18. Keely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV (1997) Cdc42 and Racl induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 390:632–636CrossRefGoogle Scholar
  19. Knupfer MM, Pulzer F, Schindler I, Hernaiz Driever P, Knupfer H, Keller E (2001) Different effects of valproic acid on proliferation and migration of malignant glioma cells in vitro. Anticancer Res 21:347–351PubMedGoogle Scholar
  20. Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschlaeger M, Dolznig H (2013) In vitro cell migration and invasion assays. Mutat Res 752:10–24CrossRefGoogle Scholar
  21. Kurzępa-Skaradzińska A, Skaradziński G, Weber-Dąbrowska B, Zaczek M, Maj T, Slawek A, Switalska M, Maciejewska M, Wietrzyk J, Rymowicz W, Gorski A (2013) Influence of bacteriophage preparations on the migration of HL-60 leukemia cells in vitro. Anticancer Res 33:1569–1574PubMedGoogle Scholar
  22. Liang CC, Park AY, Guan JL (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2:329–333CrossRefGoogle Scholar
  23. Nobes CD, Hall A (1999) Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 144:1235–1244CrossRefGoogle Scholar
  24. Poujade M, Grasland-Mongrain E, Hertzog A, Jouanneau J, Chavrier P, Ladoux B, Buguin A, Silberzan P (2007) Collective migration of an epithelial monolayer in response to a model wound. Proc Natl Acad Sci USA 104:15988–15993CrossRefGoogle Scholar
  25. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science 302:1704–1709CrossRefGoogle Scholar
  26. Roberts AW, Kim C, Zhen L, Lowe JB, Kapur R, Petryniak B, Spaetti A, Pollock JD, Borneo JB, Bradford GB, Atkinson SJ, Dinauer MC, Williams DA (1999) Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 10:183–196CrossRefGoogle Scholar
  27. Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW (2017) Image J2: ImageJ for the next generation of scientific image data. BMC Bioinform 18:529–555CrossRefGoogle Scholar
  28. Savino W, Mendes-Da-Cruz DA, Smaniotto S, Silva-Monteiro E, Villa-Verde DM (2004) Molecular mechanisms governing thymocyte migration: combined role of chemokines and extracellular matrix. J Leukoc Biol 75:951–961CrossRefGoogle Scholar
  29. Shaw LM, Rabinovitz I, Wang HH, Toker A, Mercurio AM (1997) Activation of phosphoinositide 3-OH kinase by the α6β4 integrin promotes carcinoma invasion. Cell 91:949–960CrossRefGoogle Scholar
  30. Steeg PS (2006) Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 8:895–904CrossRefGoogle Scholar
  31. Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Brocker EB, Friedl P (2003) Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160:267–277CrossRefGoogle Scholar
  32. Wyckoff JB, Pinner SE, Gschmeissner S, Condeelis JS, Sahai E (2006) ROCK- and myosin-dependent matrix deformation enables protease-independent tumor cell invasion in vivo. Curr Biol 16:1515–1523CrossRefGoogle Scholar
  33. Young JD, Lawrence AJ, MacLean AG, Leung BP, McInnes IB, Canas B, Pappin DJ, Stevenson RD (1999) Thymosin beta 4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids. Nat Med 5:1424–1427CrossRefGoogle Scholar
  34. Zantl R, Horn E (2011) Chemotaxis of slow migrating mammalian cells analyzed by video microscopy. Methods Mol Biol 769:191–203CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Morphology, Institute of Biosciences of BotucatuUniversidade Estadual Paulista “Júlio de Mesquita Filho” -UNESPBotucatuBrazil

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