This investigation reports, for the first time, the identification of TRPM2 and CD38 surface expression on human NK cell subsets in healthy participants. This paper is also the first to develop a methodology that quantifies TRPM2 and CD38 surface expression with an antibody that has not been previously applied using flow cytometry. This novel method may have significant implications for analysing TRPM2 and CD38 surface expression in vitro and may facilitate a better understanding of the role of TRPM2 and CD38 in disease pathology involving immune cells such as NK cells.
In order to characterise TRPM2 surface expression, an extracellular TRPM2 antibody was preferred to prevent non-specific binding. The predominant clonality available on the market is polyclonal intracellular TRPM2 antibodies. Intracellular TRPM2 ion channels were not investigated as cell fixation and permeabilisation provides access to intracellular antigens. As TRPM2 is also localised on intracellular compartments, such as the endoplasmic reticulum and lysosome, cell permeabilisation can enable non-specific binding and activation of these intracellular TRPM2 channels, which potentially can mediate a number of downstream signalling pathways, such as Ca2+ influx (15). Thus, a rabbit IgG polyclonal extracellular TRPM2 antibody (Thermo Fisher Scientific, USA, OST00112W) was chosen due to its ready availability and extracellular binding, specifically to the third extracellular loop of the human TRPM2 receptor.
A total of eight healthy Australian participants were age (27.50 ± 8.08) and sex-matched (Table 1). No significant differences were reported for full blood count parameters between participants (Table 2). As this project was the first to use an antibody that has only been used for western blot and immunohistochemistry, the recommended dilution series (1:300) by the manufactures’ instructions was used as a baseline. With this reference, four additional primary TRPM2 antibody dilutions (1:100, 1:50, 1:10 and 1:5) were investigated to determine the optimal primary TRPM2 antibody concentration. Additionally, two incubation periods (1 h – primary TRPM2 antibody/30 min – secondary conjugated TRPM2 antibody) and (2 h/1 h) were tested to determine the optimal incubation time for TRPM2 and TRPM2/CD38 surface binding and expression (Additional file 4: Figure S4, Additional file 5: Figure S5, Additional file 6: Figure S6, Additional file 7: Figure S7, Additional file 8: Figure S8, Additional file 9: Figure S9, Additional file 10: Figure S10, Additional file 11: Figure S11, Additional file 12: Figure S12, Additional file 13: Figure S13, Additional file 17: Figure S17, Additional file 18: Figure S18, Additional file 19: Figure S19, Additional file 20: Figure S20, Additional file 21: Figure S21, Additional file 22: Figure S22, Additional file 23: Figure S23, Additional file 24: Figure S24, Additional file 25: Figure S25, Additional file 26: Figure S26).
One limitation of the primary TRPM2 antibody was the absence of a determined antibody concentration. According to Thermo Fisher Scientific, “antibody concentrations in ascites fluid, culture supernatant and serum are not determined due to various proteins in serum which makes it impossible to acquire an accurate concentration of a specific antibody”. Due to the absence of a determined antibody concentration, a TRPM2 isotype control could not be performed. However, as the primary TRPM2 antibody contains rabbit serum, normal rabbit serum (Thermo Fisher Scientific, USA, 01–6101) was used at comparable dilutions as the primary TRPM2 antibody. This negative control was used to distinguish any non-specific binding, as well as determine an individual positive TRPM2 and TRPM2/CD38 gate for each participant (Fig. 4b, c). Additionally, an unstained tube; a secondary tube; and a FMO control (Fig. 4a) were performed for each participant to compensate any potential fluorescence spill over (Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3, Additional file 14: Figure S14, Additional file 15: Figure S15, Additional file 16: Figure S16).
On both NK subsets, a consistent pattern was observed for TRPM2 and dual surface expression with CD38. At 2 h/1 h, TRPM2 (Fig. 2a, b, p < 0.05) and TRPM2/CD38 (Fig. 3a, b, p < 0.05) surface expression significantly increased between 1:300 and 1:50 at 2 h/1 h. Additionally, a significant increase in TRPM2/CD38 expression was also observed on CD56DimCD16+ NK cells between 1:100 and 1:50 at 2 h/1 h (Fig. 3a, p < 0.05). These results indicate that 1:50 may be the optimal antibody concentration to measure TRPM2 and TRPM2/CD38 surface expression on NK cells.
The specificity of the primary TRPM2 antibody was investigated by measuring the dual surface expression of co-markers, TRPM2 and CD38, on CD56BrightCD16Dim/− and CD56DimCD16+ NK cells. Given comparable results were observed with (Fig. 3a, b, p < 0.05) and without CD38 expression (Fig. 2a, b, p < 0.05), these findings validate the specificity of the TRPM2 antibody for accurate and consistent measurement of TRPM2 surface expression.
Interestingly, a normal distribution curve was observed on both NK subsets for TRPM2 and dual expression with CD38 at 2 h/1 h. Comparatively, receptor surface expression remained relatively constant at 1 h/30 min on both NK subsets. This observation supported the significant decrease in TRPM2/CD38 surface expression from 1:50 to 1:5 on CD56BrightCD16Dim/− NK cells (Fig. 3a, p < 0.05). Importantly, this result demonstrates an inverse relationship between antibody concentration and receptor expression and highlights 1:50 as the threshold antibody dilution for TRPM2 (Fig. 1).
In contrast there was a significant increase in TRPM2 surface expression with 1:300 at 1 h/30 min (Fig. 2b, p < 0.05), but not with dual expression with CD38 (Fig. 2b, p < 0.05). This sole result revealed a difference in receptor surface expression between incubation periods. As CD56BrightCD16Dim/− NK cells are less abundant than the CD56DimCD16+ subset, the percentage of receptor expression increases with limited cells detected. Moreover, the percentage of receptor expression increases for rarer channels. Given TRP ion channels are relatively scarce, particularly on lymphocytes, a longer incubation time is required to ensure optimal binding and subsequent surface expression. Moreover, the consistent pattern with the 1:50 TRPM2 dilution on both NK subsets justified 2 h/1 h as the optimal incubation period to ensure a sufficient timeframe for maximal antibody binding and surface expression.
Despite tested applications for western blot and immunohistochemistry assays, no additional studies have published the use of the OST00112W TRPM2 antibody. Future directions include the examination of TRPM2 and CD38 channels on additional lymphocytes, as well as investigate the manufacturer’s tested applications to further assess antibody specificity.