Voie d'immunisation et séquence d'administration de l ... - TEL

tel-00827710, version 1 - 29 May 2013
3.4 Enrichment
1. To start the enrichment protocol, resuspend labeled cells obtained in Section 3.3 (step
8) in 400 μL PB.
2. Take a 10 μL aliquot of labeled cells, and place it into 5mL FACS tubes. Complete
with 90 μL with PBS-2%FCS. This gives you your « Pre-enriched » fraction.
3. To the cells used for enrichment, add 100 μL of anti-PE microbeads (see Note 18).
4. Vortex and incubate for 20 min at 4°C in the dark.
5. Wash twice in 2 mL cold PB, spinning cells at 300g for 5 min at 4°C.
6. During the washing step, prepare MACS columns (one per Falcon 15mL tube) on a
magnet support (see Note 4). Rinse each column with PB (discard elution). Label
Falcon 15 ml tubes for collecting the flow through fraction.
7. Resuspend each sample in 1 mL PB, and load the column.
It is important to filter cells just prior to loading on the column in order to remove
any clumped cells.
8. Wait until the sample has completely passed through the column bed.
9. Add 1mL of PB to the initial Falcon 15mL tube (wash step to get every last cell).
10. Load column with this fraction.
11. Wait until the sample has completely passed through.
12. Collect first flow-through fraction and load it on the same column a second time (again,
an effort to capture all multimer labeled cells).
13. Again, wait until the sample has completely passed through the column bed.
14. Wash the column with 3x1 or 2x3 mL of PB (for MS and LS columns respectively).
15. Wait until the sample has completely passed through: the collective liquid in the
collection tube (flow through fraction) is your « Depleted fraction ».
16. Remove one column at a time. Place it in a corresponding labeled Falcon 15mL tube.
Add 2-5 mL (for MS and LS columns respectively) of PB to the upper fraction of the
column. Push the plunger using steady pressure.
17. Gently remove the plunger.
18. Add again 2-5 mL of PB to the upper fraction of the column.
19. Push the plunger. The collective liquid (total volume is 4-10 mL) is considered the
« Enriched fraction ».
20. Spin Depleted and Enriched fractions at 300g for 5 min at 4°C.
21. For the Depleted fraction (see Note 19):
a) Resuspend in 1 mL of PBS-2%FCS.
b) Aliquot 90 μL in one 5ml FACS tube and add Ab mix.
c) Incubate 20 min at 4°C in the dark.
d) Wash in 3 mL PBS-2%FCS at 300g for 5 min at 4°C.
e) Resuspend in 300 μL PBS-2%FCS.
22. For the Enriched fraction, you can either continue with ICS (proceed to Section 3.7) or
prepare your samples for flow cytometry analysis on Section 3.5:
a) Resuspend in 90 μL PBS-2%FCS.
b) Add your Ab mix directly into the Falcon 15mL tube.
c) Incubate 20 min at at 4°C in the dark.
d) Add 1 mL of PBS-2%FCS; transfer to 5mL FACS tubes.
e) Add 1 mL of PBS-2%FCS to the initial Falcon 15mL tube.
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