CHAPTER 2: General Materials and Methods
2.4 Flow Cytometry
Isolated leukocyte cell populations from whole blood (all constituents, PBMC) or cell culture (MDM, MM6) were, where appropriate, depleted of erythrocyte contamination by ACK lysis buffer and re-suspended in flow cytometry buffer (FACS buffer: 5% FBS in PBS), counted, and aliquots of between 0.2-1x106cells/condition made in flow cytometry tubes or ‘v’ shaped plates. If ACK lysis buffer were employed cells were washed twice with flow cytometry wash buffer (FACS wash: FACS buffer with 2mM EDTA).
2.4.2 Antibodies
Working dilutions of each antibody were either established previously by the host laboratory by titration and assessment of expression of cell surface markers on circulating leukocytes or taken from the manufacturers literature. The list of antibodies used, including information on the fluorescent conjugate, clone, manufacturer, concentration employed, and whether an intra [I] or extra-cellular target [I] are given in Table t2.2. All antibodies employed were directly conjugated.
Panels from this set were designed dependent upon experimental question with reference to spectral overlap and relative expression on target populations.
FluoroFinder® was employed to aid panel setup and verification.
2.4.3 Cell Surface Staining
In order to reduce nonspecific binding cells were first incubated in Human TruStain FcX (Biolegend) (15mins, 4°C) to block Fc receptors. Cells were then centrifuged (500g/3mins/4°C), washed in FACS wash, re-spun and the pellet re-suspended in the volume of FACS buffer required to render the end volume of the cell/buffer/antibody mix 100μl. Selected antibodies targeted at cell surface antigens were then added as a pre-created cocktail (‘master mix’) and kept at 4oC for 30 minutes in the dark. After incubation, 50µL wash buffer was added to the mixture before centrifugation (500g/3mins/4oC). Cells were washed twice more by adding 100µL wash buffer and repeating the spin. If intra-cellular staining was not planned cells were re-suspended in 150µL FACS Buffer and 150µL fixative (0.1% paraformaldehyde in PBS) added prior to transfer to a 1mL microtube for analysis.
Target Fluorochrome Clone Manufacturer Stock []
(µg/ml)
V
(µl/sample)
CD11b [E] PerCP-Cy5 ICRF44 Biolegend NS 1.25μl
CD14 [E] AF700 MSE2 BDPharm 500μg/ml 2.5μl
CD16 [E] APC 3G8 Biolegend 150μg/ml 1μl
CD16 [E] PE/Cy7 3G8 Biolegend 200μg/ml 1.25μl
CD33 [E] PE WM53 Serotec 1mg/ml 5μl
EP2 [I] PE Poly Abcam 100μg/ml 3μl
EP4 [I] APC Poly Abcam 100μg/ml 3μl
HLA-DR [E] APC-H7 G46-6 BDPharm NS 2.5μl
HLA-DR [E] BV421 L243 Biolegend 25μg/ml 1.25μl
CD3 [E] FITC HIT3a Biolegend 200μg/ml 1.25μl
CD19 [E] FITC HIB19 Biolegend 400μg/ml 1.25μl
CD20 [E] FITC 2H7 Biolegend 100μg/ml 1.25μl
CD56 [E] FITC MEM-188 Biolegend 400μg/ml 1.25μl
CD66b FITC G10F5 Biolegend 300μg/ml 1.25μl
CD88 [E] PE/Cy7 S5/1 Biolegend 200μg/ml 2.5μl
Table t2.2: Antibodies employed. Target designates the cellular protein to which the antibody binds. [E]/[C] denotes whether the antibodies target epitope is located extra-cellularly ([E]) or intraextra-cellularly ([C], thus requiring permeabilisation). The fluorochromes give the labels a recognisable property for flow cytometry. The clones refer to the B cell clone from which these monoclonal antibodies have been obtained. It is advisable to use only one clone throughout to avoid any differences in binding affinity and eventually output signal.
2.4.4 Permeabilisation and Intra-Cellular Staining
To achieve intra-cellular staining, cells washed of excess extra-cellular antibodies were permeabilised via re-suspension in BD Bioscience Cytofix/Cytoperm® solution (100μL) for 20mins at 4°C. Cells were subsequently washed twice with, and then re-suspended in BD Perm/Wash® solution, and fluorochrome-conjugated antibodies targeted at intra-cellular epitopes added to create a total volume of 100μL. This mixture was incubated for a further 30mins (4°C/dark). Cells were subsequently washed twice prior to re-supension in FACS buffer, transfer to a microtube and made up to a volume of 300μL.
2.4.5 Compensation and Isotype Control
Where necessary, compensation controls were established for each fluorochrome used in the antibody mix to control for spectral overlap. BD CompBeads® were employed as they bind to all antibodies with uniform efficiency. Compensation controls were prepared by mixing 60μL of positive beads, 60μL of negative beads and the appropriate volume of antibody. The mixture was incubated for 30mins (4°C/dark). Stained beads were washed three times with 1mL of FACS wash with centrifugation at 800g (5mins/4°C). Following the final wash step, supernatant was discarded and the stained bead pellet re-suspended in 300μL PBS and transferred to a FACS tube. Compensation between fluorochromes was calculated automatically by the BD FACS Diva software. A maximum tolerance of 30% spectral overlap was allowed between fluorochromes.
Isotype controls are employed to help identify and discount non-specific binding (NSB). Here either the fraction-antigen binding (Fab) portion of a fluorochrome tagged antibody binds to a low affinity, non-specific target on the cell surface (or intracellular target if membrane integrity is compromised) or the fraction crystallisable (Fc) portion binds to Fc-receptors (FcR) expressed on certain cell types. Without appropriate controls this cell may be falsely identified as positive for the surface marker associated with the fluorochrome, being indistinguishable from one expressing the target epitope and specifically bound by the antibody-fluorochrome conjugate. Isotype controls are ideally antibodies from the same species and clone, with the same heavy chain, light chain, fluorochrome and F:P ratio (fluorescent molecules per antibody) as the antibody-conjugate to be employed but targeting a protein not found on the cell surface. Cells may then be incubated with this isotype and those binding it (inevitably non-specifically) may then be excluded from analysis leaving true-positives only.
Unfortunately several problems exist with the control: availability of isotype controls for each selected antibody-fluorochrome conjugate, cross-reactivity to a similar epitope on a different antigen, cost, requirement for additional cells from often limited samples etc. These are discussed in full here289, and have lead to the adoption of alternate strategies to combat NSB. These include: titration of reagents to ensure high signal in bright populations while reducing spread in negative populations, employment of Fc-block, ensuring high and consistent cell viability (reducing
‘stickiness’ associated with necrosis/apoptosis) and use of FMOs to determine positivity. Given the complexity of the panels employed and these valid concerns a
strategy of pre-experimental optimisation, viability confirmation and Fc-block was selected as opposed to use of individual isotype controls.
2.4.6 Data Gathering and Analysis
Samples were analysed using the LSR FortessaTM flow cytometer (BD Bioscience, USA) either on the same day as staining if permeabilisation had been performed or within 48hours of fixation if not.
Data were analysed using FlowJo software (FlowJo v7.6.1, Tree Star Inc., USA). Cell populations were identified using dot plots based on size (from forward scatter, FSC), granularity (from side scatter, SSC) and fluorescence. A gating strategy was adapted from previous data obtained by the host laboratory to identify individual cell populations. Cells positive for a marker were identified by performing fluorescence-minus-one (FMO) controls, made up of cells stained using an antibody mixture containing the entire panel except for one. Populations are labelled according to relative fluorescence intensity, indicated by superscript text i.e. xhi or x+/++ for high expression and xlo or x- for low or no expression.
Identified populations are given as a percentage of the total cell number (linear scale, mean ± SD) and as absolute cell numbers (logarithmic scale, median ± interquartile range). Differences between time points, differentiation and stimulation conditions are assessed where possible by paired Student’s t tests for the percentage of total cells, or by Wilcoxon matched pairs tests for absolute cell numbers. Median fluorescence intensity (MFI) is given in arbitrary units (logarithmic scale, median ± interquartile range) as this value varies between cytometers and used settings.