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tel-00827710, version 1 - 29 May 2013
4. Add 10 mL of Mouse Pulldown Buffer (MPB), count them, spin down at 300g for 5
min at 4°C, and go to Section 3.3.
3.2 HLA typing (human)
1. Generic haplotyping of the sample can be easily achieved by flow cytometry, and is
sufficient for most multimer uses.
2. Count PBMCs and resuspend in PBS at 10 7 cells/mL.
3. Dispense 2x50 μL of this solution into 5mL FACS tubes. The remaining cells will be
spinned down (300g, 5 min, 4°) and used for multimer staining (Section 3.3).
4. Add either isotype or anti-HLA antibody titrated to the optimal concentration to each
FACS tube (optimal Cf=1/400 in our hands, meaning that you put 1 µL of a solution
diluted 1/8 in 50 μL staining volume).
5. Incubate for 15 min at 4°C in the dark.
6. Wash cells once at 300g for 5 min at 4°C, and resuspend in 100 μL of PBS.
7. Acquire these 100 μL in flow cytometry (Figure 3).
3.3 Multimer staining
1. Use cells prepared on Section 3.1 (mice) or 3.2 (human). Resuspend cells in cold
Pulldown Buffer (MPB for mice or HBP for human, hereafter referred as PB), and
dispense defined numbers of cells in Falcon 15mL tubes (one for each specificity) (see
Note 13).
2. Wash once in PB (300g, 5min, 4°), and resuspend each sample in 90 μL cold PB.
3. Add 10 μL of FcR Blocking Reagent to each tube. Vortex.
4. Incubate 10 min at 4°C.
5. Add PE MHCI multimer and APC MHCI multimer of the same specificity at the
appropriate concentration (see Notes 14 and 15, and Figure 4).
6. If needed, it is possible to multiplex the experiment (i.e. determine multiple
specificities - up to 25 - within one single tube) by preparing each specific multimer
with a unique combination of two different colors [21]. In the case you want to
simultaneously enrich your target populations with antiPE microbeads, one of these
two colors will have to be PE (see Note 16 and Figure 6A).
7. Vortex gently and incubate 30 min at 4°C (see Note 17).
8. Wash once in 2 mL PB, spinning at 300g for 5 min at 4°C.
9. If you stop here, transfer your cells into 5mL FACS tubes, spin, resuspend in 90 μL of
PBS-2%FCS, and proceed directly to flow cytometry analysis on Section 3.5.
Otherwise, you can follow the procedure by enrichment (Section 3.4).
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