Chapter 7: Materials and Methods
7.8. Flow cytometry
7.8.1. Preparation of single-cell suspensions
Lymphoid organs from relevant mice were harvested by dissection. Cell suspensions from the isolated lymphoid organs were obtained by passing the organs through a 70µm nylon mesh strainer (Becton Dickinson, Oxford, UK) using the base of a 1ml syringe plunger. The cells were washed twice with PBS and spinning at 1,500rpm. Where necessary, blood, thymus and spleen cells were depleted of RBC using 1ml of RBC lysis buffer (Qiagen, UK) and incubated for 10 minutes at room temperature. Cells were counted after washing, as described in Section 7.4, to prepare a cell concentration of 107 cells per ml re-suspended in PBS.
7.8.2. Cell surface staining
For each cell staining condition, 0.5µl of the necessary fluorochrome-conjugated antibody (1:200 dilution) was added to a suspension of 106 cells in 100µl of PBS. The samples were incubated in the dark at room temperature for 20 minutes. This was followed by washing twice with 3ml of PBS and spinning at 1,500rpm for 5 minutes. The supernatant was discarded and the cells were re-suspended
157 in a final volume of 400µl of PBS. The samples were kept at 4℃ before acquisition. Acquisition of flow cytometry data was performed on a FACSCalibur™ cytometer using the CellQuest™ software (BD Biosciences). At least 50,000 and 100,000 cells were collected for thymus and spleen analyses respectively. Cells from WT C57BL/6 mice were used as single-stained controls to set up compensation settings. Data analysis was performed using the FlowJo version 10 software (FlowJo, LLC, USA). The full list of antibodies is summarised below (Table 7.5). All antibodies were purchased either from BioLegend, UK or eBioscience, UK.
Table 7.5. List of monoclonal antibodies.
Antigen Fluorochrome Origin Clone
FoxP3 (Intracellular) PE Rat NRRF-30
TCRβ (C domain) PE, APC Armenian hamster H57-597
Vβ6 TCR PE Rat RR4-7
Vβ8.3 TCR PE Mouse 8C1
CD3ε PE Armenian hamster 145-2C11
CD4 PerCP, APC Rat RM4-5
CD5 PE Rat 53-7.3
CD8α PerCP Rat 53-6.7
CD8β PerCP Rat YTS156.7.7
CD69 PE Armenian hamster H1.2F3
7.8.3. MHC Dextramer™ staining
Cells from the MataHari TCR (CDR3β diversifying) retrogenic mice were stained with PE-conjugated CD8+ MataHari-specific Uty peptide (WMHHNMDLI) in the context of H2-Db MHC Dextramer™ (Immudex, Denmark). Staining of cells was performed according to the manufacturer’s protocol. In brief, 1-3 x 106 cells were transferred into polystyrene tube and 2ml PBS containing 5% FCS at pH7.4 added before centrifugation at 300g for 5 minutes. The supernatant was removed and the cells were re-suspended in a total volume of 50µl of PBS (5% FCS, pH7.4). 10µl of the MHC Dextramer™ was added and to this suspension and incubated in the dark for 10 minutes. Additional antibodies (e.g. anti-CD4 and anti-CD8 Ab) were added afterwards and incubated in the dark at 2-8℃ for 20 minutes. This was followed by washing twice with 2ml PBS (5% FCS, pH7.4) and spinning at 300g for 5 minutes. The supernatant was discarded and the cells were re-suspended in 400µl of PBS and stored at 2-8℃ in the dark until analysis.
158 7.8.4. Multimer staining
Cells from the Marilyn TCR (CDR3β diversifying) retrogenic mice were stained with PE-conjugated CD4+ Marilyn-specific Dby peptide fragment (NAGFNSNRANSSRSS) bound to an H2-Ab multimer (TCMetrix, Switzerland). Multimer staining was performed following the recommended staining of CD4+ T cells. Briefly, 106 cells were re-suspended in 50µl of PBS containing 0.5% BSA, 5mM EDTA and 0.02% sodium azide. The cell suspension was incubated with 10µl of the multimer (50µg/ml concentration), along with other antibodies (anti-CD4, anti-CD8 Ab), at 37℃ in the dark for 30-60 minutes. The cells were then washed twice with 2ml of PBS (0.5% BSA, 5mM EDTA and 0.02% sodium azide) before a final wash with 2ml PBS. The supernatant was removed before re-suspending the cells in 400µl of PBS. Cells were stored at 4℃ in the dark before flow cytometry analysis.
7.8.5. Fluorescence-activated cell sorting (FACS)
For purification of specific splenic T cell subsets from retrogenic mice, single-cell suspensions were prepared as in Section 7.8.1. These cells were stained with a combination of anti-Vβ6, anti-CD4 and anti-CD8 Ab for Marilyn TCR (CDR3β diversifying) or anti-Vβ8.3, anti-CD4 and anti-CD8 Ab for MataHari TCR (CDR3β diversifying) as described in Section 7.8.2. The samples were re-suspended in sort buffer at a concentration of 20 million cells per ml and filtered through 35µM nylon mesh. The FACSAriaII™ flow cytometer at the MRC Clinical Sciences Centre Flow Cytometry Facility in Hammersmith Hospital was used for sorting cells. For Marilyn TCR (CDR3β diversifying) samples, T cells were separated based on CD4+Vβ6+ and CD4+Vβ6- gating. Conversely, MataHari TCR (CDR3β diversifying) T cells were divided based on CD4+Vβ8.3+, CD8+Vβ8.3+ CD8+Vβ8.3- gating. Sorted cells were collected into collection buffer, centrifuged at 1,500rpm for 5 minutes and supernatant removed before RNA extraction (Section 7.6.7).
7.8.6. Intracellular FoxP3 staining
Cells from the retrogenic mice expressing the Vα-Cβ fusion TCR chain were first stained with anti-CD4 and anti-CD8 Ab as described in Section 7.8.2. After the last wash, the supernatant was removed and sample vortexed before adding 1ml of FoxP3 Fixation/Permeabilisation working solution. The cell suspension was then incubated at 4℃ for 60 minutes in the dark and washed twice with 2ml of 1X Permeabilisation buffer. Following re-suspension in 100µl 1X Permeabilisation buffer, the cells were labelled with PE-conjugated anti-FoxP3 (intracellular) Ab and incubated at room temperature for 30
159 minutes. The stained cells were washed twice with 2ml of 1X Permeabilisation buffer before re- suspension in 400µl of PBS. Cells were stored at 4℃ in the dark before data acquisition. Splenocyte suspensions from WT C57BL/6 mice which have been treated with the FoxP3 Fixation/Permeabilisation working solution and either stained or unstained with the anti-FoxP3 Ab were used as the positive and negative controls respectively.