From OpenWetWare
Jump to navigationJump to search

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:

  1. 65x106 PBMC for cell culture.
  2. 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.
  3. 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:

  1. 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.
  2. 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.
  3. Incubate for ~18h total.
  4. Add 20 μL CD154-PE antibody to all wells for the last ~3h of culture, and mix with P1000 pipette.
  5. 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
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.


discuss this protocol