Ex vivo coculture assay T cells – CD11b cells

-  Media : RPMI 10%FBS, PS, beta-mercaptoethanol, NEAA

-  Control wells: unstained, LD only, T cells alone, CD11b alone

-  Perform assay with triplicate wells

I. CD11b isolation from tumor and spleen

• Harvest the tumors using the protocol for tumor cell isolation from biopsy or smash the spleen (using the back of a syringe) and filter using a 40um filter (no red blood cell lysis needed when processed for cell isolation)
• Centrifuge at 1500rpm for 5 min
• Resuspend in sterile FACS buffer
• Count the cells and follow the Mouse CD11b Positive Selection Kit II instruction
• After isolation count the cells and resuspend in RPMI 10%FBS, PS, B-merc, NEAA at 2-2.5.10⁶ cells/mL in order to have 200-250 000 cells/100uL

II. T cells isolation: CD4 and cd8 T cells

(From the same spleen suspension)

• Count the cells and follow the EasySep™ Mouse CD8+ T or CD4+ T Cell Isolation Kit
• Centrifuge at 1500rpm for 5 min
• Resuspend in sterile FACS buffer
• Count the cells
• CFSE staining:
  • Resuspend in PBS at 1.10⁷ cells/mL and add CFSE at 1uM
  • 6min RT
  • Quench labeling with RPMI 10%FBS, PS
• Centrifuge at 1500rpm for 5 min (green visible pellet)
• Count the cells and resuspend in RPMI 10%FBS, PS, B-mercapto, NEAA at 2-2.5.10⁶ cells/mL in order to have 200-250 000 cells/100uL
• After isolation count the cells and resuspend at 2-2.5.10⁶ cells/mL in order to have 200-250 000 cells/100uL
• Add CD3/CD28 dynabeads to activate the T cells

III. Coculture

• In a Cellstar Grenier flat bottom 96 wells plate, add the CD11b cell suspension and the T cells (supplemented in CD3/CD28 dynabeads) to the plate (ratio 1:1)
• Leave for 48-72h in the incubator

IV. FACS staining

• Harvest the supernatant and non-attached cells and put in a new 96 round bottom plate
• Centrifuge 2 min 1 800 rpm RT
• Wash with PBS the flat bottom plate and add into the new plate (if you want to harvest the supernatant, do it before adding the PBS)
• Add 50uL/well of trypsin and put at 37 to detach cells left in the flat bottom plate
• Add 50uL/well of FACS buffer on top of the cells and trypsin-
• In the meantime, centrifuge the plate with supernatant and PBS (If you want to collect the supernatant for ELISA or CBA do not add the PBS, collect the supernatant after centrifugation and put at -80, then proceed with the addition of PBS)
• Transfer the cells with trypsin and FACS buffer in the new plate and add 100uL of PBS
• Centrifuge 2 min 1 800 rpm RT and remove the supernatant
• Perform LD staining (LIVE/DEAD Fixable Violet) diluted in PBS (1/1000) 15 min on ice
• Add 100uL of FACS buffer
• Centrifuge 2 min 1 800 rpm RT and remove the supernatant
• Resuspend in FACS buffer

V. FACS analysis

• Go to FACS and use:
  • Laser 488 620/20 - Gallios (FL9) : LD violet
  • Laser 488 525/40 - Gallios (FL1) : CFSE
  • (FITC)

Annexes:

• Product references

        EasySep™ Mouse CD11b Positive Selection Kit II   StemCell # 18970

        EasySep™ Mouse CD8+ T Cell Isolation Kit            StemCell # 19853

        EasySep™ Mouse CD4+ T Cell Isolation Kit            StemCell # 19852

        CFSE                                                                      Molecular Probes #C-1157

        LIVE/DEAD Fixable Violet Dead cell stain kit       ThermoFisher scientific (Life Technologies) # L34964

        CD3/CD28 dynabeads                                             Gibco #11456D

        NEAA (100X)                                                            Gibco #1140-035

        beta-mercaptoethanol (1000X)                               Gibco #21985-023