The preliminary experiments stage of detecting TRPV1 using flow cytometry was executed at the Launceston General Hospital (LGH) FACSCalibur™ flow cytometer (Becton Dickenson Biosciences, San Jose, USA). Thirty one experiments were conducted in the preliminary stage for method optimisation.
Immunophenotyping
All fluorochrome-conjugated CD markers are purchased from Becton Dickenson (San Jose, USA) unless other wise stated. A combination of peridinin chlorophyll protein (PerCP), fluorescein isothiocyanate (FITC), allophycocyanin (APC), and phycoerythrin (PE) conjugated antibodies were selected. CD3-APC (T-cells), CD14- APC (monocytes), CD19-PE (B-Cells) and CD56-PE (natural killer cells) were used for leukocyte immunophenotyping (surface staining) using the FACSCalibur™.
Flow Cytometer Calibration
The FACSCalibur™ cytometer was set to lyse/wash method and the calibration was performed using BD Calibrite™ beads (Becton Dickenson Biosciences, San Jose, USA).
Electronic Optimisation
Four tubes each containing 1 x 106 white blood cells, obtained from normal subject, were stained with CD45-FITC, CD3-PE, CD45-PerCP, or CD45-APC, separately,
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and two tubes each containing 1 x 106 white blood cells were subjected to sample processing, with equivalent volumes of PBS in place of the CD markers (unstained cells) for electronic optimisation. All incubations were performed at ambient temperature, unless otherwise stated. Cells were stained with CD markers for 10 minutes, in the dark. Cells were then incubated in 2 mL FACS Lyse solution (BD Biosciences, San Jose, U.S.A) for 10 min, in the dark. Cells were then centrifuged at 300 x g for 5 min, washed in 2 mL PBSA, and re-centrifuged at 300 x g for 3 min.
Forward and Side Scatter Optimisation
Forward scatter (FSC) amplification (amp) gain and side scatter (SSC) voltage were adjusted to position the three main leukocyte subpopulations; lymphocytes, monocytes and granulocytes on the linear scale (Figure I). FSC threshold was adjusted to minimise debris appearing on the scale. FSC and SSC were optimised for all samples under all conditions (Figure II).
Figure I: FSC and SSC electronic optimisation for fixed and permeabilised cells using FACSCalibur™. A successful optimisation of FSC and SSC, leads to a clear distinction between the three main leukocyte populations, lymphocytes, monocytes, granulocytes and the exclusion of debris.
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PMT Voltage Optimisation
Photomultiplier tube (PMT) voltage optimisation was performed to separate signal (positive) from noise (negative). PMT voltages were optimised before the compensation optimisation step, using two-three drops of unstained cells in each of the single stained samples (Maecker and Trotter 2006). Subsequently, the sample was placed on the FACSCalibur™ sample injection port (SIP). Data acquisition was started and the related detector voltages were adjusted during the live set-up phase of data acquisition. The PMT voltages were adjusted to position the unstained negative cells within the first decade of the histogram. However, the positive cells were positioned within the third decade (Maecker and Trotter 2006).
Figure II: Example of optimised FACSCalibur™ settings for lyse/wash method of a normal blood sample. Detectors are the channels in which each fluorochrome is detected; voltages are the PMT values for each channel; threshold is the value where debris was excluded, and compensation matrix showing calculated percentages of corrected spillover of one channel into the other.
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Fluorescence Compensation
Compensation optimisation step was performed to eliminate fluorescence spectral overlap. For the compensation, single tubes each containing 10µL of each CD45- FITC, CD45-PerCP, CD3-PE and CD45-APC antibodies, separately, for electronic optimisation (Maecker and Trotter 2006). Unstained cells (negative population) were added to each of the single stained tubes. Compensation optimisation was performed during the set-up phase of live data acquisition after setting up the PMT voltage (Figure III). Cellular autofluorescence was also investigated, and the data showed negative cell populations located within the first quadrant, indicating minimal autofluorescence of the target cells under test conditions (Figure III, C-E).
Figure III: Example of compensation optimisation. (A) Data demonstrate PerCP voltage before optimisation, (B) PerCP voltage after electronic optimisation. CD45-PerCP negative population (A1 and B1) positioned within first decade, and CD45-PerCP positive population from second to third decade have increased with optimisation (A2 and B2), (C) Example of over-compensation, (D) under-compensation, (E) Properly compensated data.
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Fixation and Permeabilisation
Fixation and permeabilisation procedures were required to allow the TRPV1 primary antibody to bind to its intracellular epitope. Two commercially available kits CALTAG™ (Life Technologies, Grand Island, USA) and Cytofix/CytopermTM (Becton Dickenson, San Jose, USA) were investigated to determine the staining patterns of TRPV1. The fixation/permeabilisation protocols for both kits were applied according to suppliers’ instructions, with slight modifications including staining the CD markers for 10 min and centrifuging cells at 300 x g. Cells were fixed, permeabilised and washed using a saponin-based wash reagent to ensure cells permeabilised as the permeabilisation step is reversal. There was no significant difference in TRPV1 signal using either the CALTAG™ (Life Technologies, Grand Island, USA) and Cytofix/CytopermTM kits (Becton Dickenson, San Jose, USA) (Figure IV).
Secondary Antibody Titration
Secondary antibody optimisation involved titration studies for both FITC- and Alexa Flour®488-conjugated polyclonal goat anti-rabbit secondary antibodies (Figure V). The variation between Alexa Fluor®488 background staining was observed at
Figure IV: Staining patterns for two fixation/permeabilisation kits tested (A) CALTAG™ kit and (B) BD Cytofix/Cytoperm™ kit. Results demonstrate same efficiency of both kits.
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dilutions ranging from 1:50 to 1:250 (Figure V, B). Therefore, optimal dilution for goat anti-rabbit- Alexa Fluor®488- conjugated secondary antibody was 1:50 and 1:25 for FITC-conjugated secondary antibodies. Alexa Fluor®488 as a fluorochrome looks brighter than FITC, however separation between signal and noise was similar for both.
Figure V: FITC- and Alexa Fluor®488- conjugatedsecondary antibody
dilution study using the FACSCalibur™. (A) Secondary antibody alone titration for FITC, and(B) Alexa Fluor®488: an increase in TRPV1 signal
with decreasing the secondary antibody titre was noticed. The brightest signal was obtained using 1:50 dilution. (C) Optimised working dilutions with (green) and without (blue) the Santa Cruz anti-TRPV1 antibody for FITC and (D) Alexa Flour®488. Both secondary antibodies demonstrated
similar staining patterns between secondary alone control (Blue) and TRPV1 signal (Green).
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Blocking Optimisation
The following general blocking reagents were tested: human AB serum (obtained from healthy AB blood group donor), and FcR blocking reagent (Miltenyi Biotechnology, Cologne, Germany). Better staining pattern was observed using Fc- receptor blocking compared to the human AB serum (Figure VI).
Figure VI: Blocking optimisation using FACSCalibur™ cytometer on anti-TRPV1 antibody (Santa Cruz Biotechnologies). Results demonstrate better staining pattern for (B) Fc-receptor blocking compared to (A) human AB serum. (C) a significant decrease in signal was observed when using a combination of both.
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Primary antibody dilution
A dilution study for Santa Cruz Biotechnology anti-TRPV1 antibody was performed to optimise the TRPV1 signal, by incubating one million cells with 0.1 to 2 µg of the primary antibody diluted 1:20 (Santa Cruz Biotechnologies, CA, USA). There was a direct correlation between the amount of primary antibody added and the TRPV1 signal, with optimum TRPV1 antibody quantity of 1µg (Figure VII). Still, no separation between isotype control and this anti-TRPV1 antibody; as similar or weaker signal than the isotype control was detected (Figure VIII).
Figure VII: TRPV1 primary antibody optimisation (Santa Cruz Biotechnologies, CA, USA). (A) Results demonstrate increase in TRPV1 signal with increasing quantity of TRPV1 primary antibody, (B) TRPV1 signal comparing to noise.
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Data Acquisition & Analysis
Data acquisition and analysis were completed using the FACSCalibur™ flow cytometer and Cell QuestPro software (version 5.1) (Becton Dickenson Biosciences, San Jose, USA). 10,000 events were acquired for each sample. Fluorescence intensity is expressed in arbitrary units on a logarithmic scale. Final analysis was performed using FlowJo (version 7/9, Oregon, U.S.A.).
Figure VIII: TRPV1 signal compared to isotype control in human leukocyte subpopulations. Data demonstrate staining pattern for isotype control (Santa Cruz biotechnology, USA) compared with TRPV1 signal (Santa Cruz biotechnology, USA) and secondary antibody only control (goat anti-rabbit-FITC, Santa Cruz biotechnology, USA) for (A) Monocytes, (B) T cells, (C) B Cells and (D) natural killer cells using FACSCalibur™.
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