Shreffler:AADCRC

Handling of blood samples of adult PNOIT study (U19, AADCRC project 2)

These are whole blood samples of ~450 ml.
Set aside 2 ml of blood for basophil test.
Use 200 μL of blood for DNA isolation.
Put the blood in 50 ml tubes and spin at 800g, 15 min. Harvest all the plasma, put in five 5 ml polypropylene tubes (4 ml/tube) and the rest in 50 ml tubes.
Add equal volume of PBS, resuspend, do Ficoll separation, and isolate PBMC, per the usual protocol. Resuspend PBMC in 1 ml PBS and count.

Distribution of the PBMC sample:

65x10⁶ PBMC for cell culture.
200x10⁶ PBMC for positive isolation of CD4+ cells and CD19+ cells. Give negative fraction to David Alvarez.
- Give 30x10⁶ CD4+ cells to David, cryopreserve the rest, in two vials.
- Cryopreserve all CD19+ cells, in two vials.
Cryopreserve rest of the PBMC (~200x10⁶).
- Flow analysis by Sanofi (10x10⁶ in one vial).
- Flow analysis by Patrick Brennan (10x10⁶ in one vial).
- Rest in aliquots of 30x10⁶ (~6 vials).

Culture of PBMC:

Culture cells in 24-wells plate, 3 wells per variable, 5x10⁶ cells/well, in 1 ml AIM-V medium per well. Variables are Unstim, Golden PE, anti-CD3/CD28, and Ara h 1/Ara h 2, so 4x3=12 wells total. Make suspension of 65x10⁶ PBMC in 13 ml medium, then add 1 ml per well.
Add 16 μL Golden PE (100 ug/ml), 6 μL anti-CD3/CD28 beads (1:20 bead-to-cell ratio), or 30 μL Ara h 1/136 μL Ara h 2 (both 50 ug/ml) in the appropriate wells, and mix with P1000 pipette.
Incubate for ~18h total.
Add 20 μL CD154-PE antibody to all wells for the last ~3h of culture, and mix with P1000 pipette.
Harvest cells, resuspend in 150 μL staining buffer, and stain with the following panel for sorting. Use the indicated amounts per tube (~15x10⁶ cells). Remember to prepare compensation controls, and FMO control for CD69.

Staining cocktail:

Live/dead-violet 3 μL (do not add live/dead-violet to cells cultured with Ara h 1/Ara h 2).

CD3-AF700 8 μL

CD4-APCCy7 8 μL

CD45RA-FITC 30 μL

CD154-PE 30 μL

CD69-AF647 8 μL

Flow cytometry:

Gate on forward/side scatter, then live CD3+ cells, then CD3+CD4+ cells, then CD4+CD45RA- cells.

For the Unstim, Golden PE, and anti-CD3/CD28 cultures, sort CD154+CD69+/- cells, CD154-CD69+ cells, and CD154-CD69- cells (3 tubes total). Spin down the cells, lyse in buffer RLT + b-ME and store in -80C freezer.
For the Ara h 1/Ara h 2 culture, sort CD154+ and CD69+ cells (in one tube), and CD154-CD69- cells (2 tubes total). Then spin down and cryopreserve in FBS + 10% DMSO.

Discussion