Identification and characterisation of transient receptor potential melastatin 2 and CD38 channels on natural killer cells using the novel application of flow cytometry
Natural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/− and CD56DimCD16+ NK cells. Intracellular calcium (Ca2+) is fundamental to regulate a number of intracellular signalling pathways and functions in NK cells, which are essential in mediating their natural cytotoxic function. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable non-selective cation channel that possesses a critical role in calcium-dependent cell signalling to maintain cellular homeostasis. TRPM2 and CD38 protein surface expression has yet to be determined on NK cells using flow cytometry. Characterisation of TRPM2 has been previously identified by in vivo models, primarily using methods such as genetic remodification, immunohistochemistry and whole cell electrophysiology. The aim of this study was to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously applied using flow cytometry.
At 2 h/1 h, TRPM2 (Fig. 2 A, 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. TRPM2/CD38 surface expression furthermore increased between 1:100 and 1:50 at 2 h/1 h (Fig. 3A, p < 0.05). Interestingly, TRPM2/CD38 surface expression significantly decreased from 1:50 to 1:5 on CD56BrightCD16Dim/− NK cells. These consistent findings highlight that 1:50 is the optimal antibody dilution and threshold to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. 2 h/1 h was determined as the optimal incubation period to ensure a sufficient timeframe for maximal antibody binding and surface expression.
For the first time, we describe an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cells in healthy participants. Finally, using an antibody that has not been previously applied in flow cytometry, we determined an antibody concentration and incubation time that is robust, rapid and sensitive for the application of flow cytometry.
KeywordsAntibody Flow cytometry Natural killer cells Transient receptor potential Melastatin 2
Cyclic adenosine disphosphate-ribose
Fluorescence minus one
Nicotinic acid adenine dinucleotide phosphate
National Centre of Neuroimmunology and Emerging Diseases
Peripheral blood mononuclear cells
Standard error of the mean
Transient Receptor Potential Melastatin 2
Natural Killer (NK) cells are effector lymphocytes of the innate immune system found in peripheral blood, bone marrow, spleen, and lymph nodes. In peripheral blood, NK cells represent 15% of lymphocytes and are phenotypically distinguished by the surface expression of CD56 (neural cell-adhesion molecule) and CD16 (FcγIII receptor, the low affinity receptor of IgG) receptors. Thereby, NK cells are subclassed into CD56BrightCD16Dim/− and CD56DimCD16+ NK cells which represent respectively 10 and 90% of NK cells in peripheral blood . NK cells have diverse biological functions, which include recognizing and killing virally infected or transformed cells. The former NK population is primarily involved in immunosurveillance and cytokine production, whereas the latter are cytotoxic and kill infected, tumour or ‘missing self’ cells . Intracellular calcium (Ca2+) mobilisation is required to regulate a number of intracellular signalling pathways in NK cells, such as the antibody dependent cellular cytotoxicity (ADCC) or mitogen-activated protein kinase pathway, which are essential for the development of immune synapse formation, cytokine production and cytotoxic activity . Intracellular Ca2+ is also required for the target cell adhesion, granule polarization and degranulation, all of which are necessary for NK cells to mediate their natural cytotoxicity [1, 3].
Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable nonselective cation channel that is characterised with a unique C-terminal ADP-ribose (ADPR) pyrophosphate domain . TRPM2 is synergistically activated by intracellular ADPR and Ca2+ within the plasma membrane and/or lysosomal compartments. Binding of ADPR to TRPM2 opens the channel and allows the permeation of sodium (Na2+), potassium (K+) and Ca2+ into the cell and hydrolysis of ADPR to ribose 5-phosphate and adenosine monophosphate (AMP) . Previous investigations have shown that TRPM2 mediates a novel anti-tumour mechanism in NK cells in synergy with CD38, a multifunctional ectoenzyme using Nicotinamide adenine dinucleotide (NAD+) as a substrate to catalyse the production of ADPR, cyclic ADPR (cADPR) and Nicotinic acid adenine dinucleotide phosphate (NAADP) . Rah et al. (2016) demonstrated that CD38 facilitates the production of ADPR, which in turn mobilizes intracellular Ca2+ and can activate TRPM2 resulting in cytolytic degranulation and antitumor activity of NK cells .
Investigation of TRP ion channel expression on lymphocytes has been quite limited due to methodology difficulties as TRP channels are relatively low in abundance and there is limited availability of specific and high-affinity antibodies. Characterisation of TRPM2 has been predominantly investigated with in vivo models accompanied by genetic remodification [6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16], western blot [6, 13, 17], immunohistochemistry [6, 12, 15], polymerase chain reaction [10, 11, 12, 13, 15, 16, 17, 18], and whole cell-electrophysiology [8, 9, 10, 11, 13, 18] methods. In vitro investigations of TRPM2 have furthermore been examined on cell lines [10, 17, 18, 19, 20], neurons [21, 22], and immune cells [10, 11, 12, 13, 16].
TRPM3 surface expression on CD56BrightCD16Dim/− and CD56DimCD16+ NK cells has been identified on healthy participants by flow cytometry [23, 24]. Flow cytometry has been the preferred technology for determining and quantifying homogenous cell subsets  due to its single-cell levelled analysis for multiple characteristics, such as cellular features, organelles, and structural components . This sensitive and specific feature enables prompt and accurate quantification, analytical precision, superior throughput, and reproducibility , all of which are advantageous for unique and rare cell populations, such as NK cells. Current flow cytometer technologies can detect up to eighteen colours in one flow assay. Thus, the scientific prospects not only lie in biomedical research, but also for clinical applications of diagnostic value .
Currently, there are no in vitro models that have characterised endogenous TRPM2 and CD38 surface expression on human NK cells. Thus, the aim of this present study was to develop a methodology to characterise TRPM2 and CD38 surface expression on human NK cells using flow cytometry. This investigation may facilitate a better understanding of the role of TRPM2 and CD38 in disease pathology involving immune cells such as NK cells.
Immunophenotype of TRPM2 and CD38 receptors on NK cell subsets by flow cytometry
CD3−/CD56+ NK cells were sorted into CD56DimCD16/+ and CD56BrightCD16Dim/− NK cell subsets using CD56 (Pe-Cy7) and CD16 (BV650). Five antibody controls were performed to determine an individualised positive TRPM2 and TRPM2/CD38 gate for each participant. Antibody controls included an unstained tube (unlabelled NK cells); secondary tube (conjugated secondary antibody FITC); and a FMO tube (CD3, CD56, CD16 and CD38). (b) Normal rabbit serum was used at comparable dilutions as the primary TRPM2 antibody to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. (c) Normalised TRPM2 and TRPM2/CD38 surface expression was calculated by compensating the percentage of fluorescence spill over into the B525_50 (TRPM2) and V525_50 (CD38) detectors from the TRPM2 antibody stained tube on both NK subsets.
Flow cytometry gating strategy of TRPM2 and CD38 using primary TRPM2 antibody (1:50) at 2 h/1 h
TRPM2 surface expression on natural killer cell subsets in healthy participants
Dual identification of TRPM2 and CD38 surface expression on natural killer cell subsets
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.
Demographic results of healthy participants
27.50 ± 8.08
Male (n = 4)
Female (n = 4)
Full blood count parameters of healthy participants
Healthy Male Participants
Healthy Female Participants
White Cell count (× 109/L)
5.57 ± 0.41
6.14 ± 0.35
Neutrophils (× 109/L)
3.27 ± 0.30
3.76 ± 0.20
Lymphocytes (× 109/L)
1.58 ± 0.14
1.55 ± 0.29
Monocytes (× 109/L)
0.43 ± 0.03
0.51 ± 0.04
Eosinophils (× 109/L)
0.24 ± 0.05
1.15 ± 0.10
Basophils (× 109/L)
0.06 ± 0.01
0.08 ± 0.20
Platelets (× 109/L)
2.48 ± 0.72
2.32 ± 1.54
Haemoglobin (× 109/L)
142.10 ± 3.99
148.56 ± 4.50
Red Cell count (× 1012/L)
4.89 ± 0.15
4.90 ± 0.16
MCV (× 109/L)
88.10 ± 1.75
83.29 ± 1.81
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.
This novel methodology is the first to identify and characterise TRPM2 and TRPM2/CD38 surface expression on human NK cells in healthy participants. This pilot investigation is also the first to use a TRPM2 antibody that has not been previously applied in flow cytometry, as well as determine the optimal primary TRPM2 antibody dilution and incubation time. This method provides an in vitro alternative using flow cytometry to characterise TRPM2 in a rapid, robust and sensitive fashion. This pilot investigation provides insight for possible improvement in antibody design to facilitate a more accurate assessment of TRPM2 and CD38 surface expression.
From 150 screened Australian participants, ten healthy participants were selected for this pilot investigation. Two participants were excluded due to outlier values during data analysis. Participants were sourced from the National Centre of Neuroimmunology and Emerging Diseases (NCNED) database for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) between November and December 2018. Participants were excluded if they were pregnant or breastfeeding, or reported a previous history of smoking, alcohol abuse or chronic illness (for example, autoimmune diseases, cardiac diseases and primary psychological disorders). Participants donated 85 ml of whole blood in ethylendiaminetetraacetic acid (EDTA) tubes between 8:30 am and 10:00 am on the Gold Coast. All healthy participants provided written consent and the study was approved by the Griffith University Human Research Ethics Committee (HREC/15/QGC/63).
Peripheral blood mononuclear cell isolation and natural killer cell isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by centrifugation over a density gradient medium (Ficoll-Paque Premium; GE Healthcare, Uppsala, Sweden) to separate granulocytes as previously described [27, 28]. PBMCs were stained with trypan blue stain (Invitrogen, Carlsbad, CA) to determine total cell count and cell viability and adjusted to a final concentration of 5 × 107 cells/ml. NK cells were isolated from PBMCs using an EasySep Negative Human NK Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada) as previously described [27, 28].
TRPM2 Immunophenotyping assay
Two incubation periods were performed for the primary and secondary TRPM2 antibodies (1 h and 30 min vs. 2 h and 1 h) at 4 °C in the dark. Labelled cells were washed with stain buffer (BD Biosciences, New Jersey, USA) and centrifuged at 350 g for 5 min. Supernatant was removed and cells were incubated with a secondary Goat F(ab) Anti-Rabbit IgG H&L Fluorescein isothiocyanate (FITC) (1:500, Abcam, UK, ab7050) in 200 μl for 1 h/30 min. Cells were washed and stained with 5 μl of 7-AAD (BD Bioscience, New Jersey, USA) to measure cell viability. Cells were resuspended in 200 μl of stain buffer (BD Bioscience, New Jersey, USA) and acquired at 10,000 events using the LSRFortessa X-20.
LSR Fortessa X-20 flow cytometry analysis
Lymphocyte populations were identified using forward and side scatter dot plots. Exclusions were CD3+ cells and only CD3− lymphocytes were further used to characterise NK cells by CD56. CD3−/CD56+ NK cells were sorted into CD56BrightCD16Dim/− and CD56DimCD16+ NK cell subsets using CD56 and CD16. TRPM2 and CD38 surface expression was measured on CD56BrightCD16Dim/− and CD56DimCD16+ NK cell populations as percentage of parent cells (%).
Pilot data from this investigation were analysed using SPSS version 24 (IBM Corp, Version 24, Armonk, NY, USA) and GraphPad Prism, version 7 (GraphPad Software Inc., Version 7, La Jolla, CA, USA). Shapiro-Wilk normality tests were conducted to determine the distribution of data, in addition to skewness and kurtosis tests to determine data normality. The independent Mann–Whitney U test was performed to determine the statistical significance between groups in TRPM2 parameters on NK cells. Conversely, the Kruskal Wallis H test was used to determine significance in TRPM2 and CD38 surface expression within groups. Significance was set at p < 0.05 and the data are presented as mean ± standard error of the mean unless otherwise stated.
All the authors undertook the study design and collection, analysis, interpretation of data and in writing the manuscript. Funding bodies only provided financial support towards this study. Funding bodies included: the Stafford Fox Medical Research Foundation, McCusker Charitable Foundation, Buxton Foundation, Henty Community, Henty Lions Club, Mason Foundation, Mr. Douglas Stutt, Blake Beckett Foundation, Alison Hunter Memorial Foundation and the Change for ME Charity. Funding bodies.
Availability of data and materials
The datasets generated and/or analysed during the current study are not publicly available due to confidentiality agreements but are available from the corresponding author on reasonable request.
CB performed the experiment. HC, DS and SMG participated in study coordination and contributed substantially reviewing the draft manuscript. CB performed the data analysis. CB interpreted the data analysis and drafted the manuscript. CB and SMG designed the study. All authors reviewed and approved the final version of the manuscript.
Ethics approval and consent to participate
All participants provided written consent and the study was approved by the Griffith University Human Research Ethics Committee (HREC/15/QGC/63).
Consent for publication
The authors declare that they have no competing interests.
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