Method for FTOC
Prepare DeGuo plates
- 450 mL RPMI
- 50 mL FCS
- 100uL each pen/strep
- 5 mL Jungle Juice (filtered)
- 10mM HEPES
- 5mg/L Folic Acid
- 2g/L glucose
- 2ME if needed
- Take 50 ml of above. Add 0.018g 2-deguo (filtered) (Sigma,
Cat. no: D-0901 for 250mg)
- Add 2mls per small petri dish.
- Control plate has 2ml normal medium.
- Place 2x gelfoams (soaked, cut in half).
- Place filter disc (dipped in the medium) on each gelfoam.
- Place in 37oC, 5% CO2 incubator O/N to ensure sterility.
- Prepare lobes.
- Make sure they’re fairly clean.
- Remember: when removing lobes from torso, make sure it is
- Place 5-8 lobes/disc.
- Incubate as before.
Wash every 2-3 days (use pipette with medium from
petri dishes and do GENTLY!
Performed 6 days after Deguo culture.
NB: Wash lobes in 50 mls of RPMI (large petri dish -
alright to leave on discs) for 2 x 2hr washes. DO FIRST THING!!
- Remove livers.
- Push through a sterile sieve (100um).
- Centrifuge (1500rpm, 5mins, 10ml tube).
- Resuspend in 10mls.
- Layer 5mls over 5mls Ficoll-Paque (sterile).
- Centrifuge 3000rpm, 35mins (Carbone lab).
- Remove lymphocyte layer, pool, spin, resuspend and count
- Want 5 x 10^4 cells/well.
- 1 lobe per well.
- Therefore, no. of wells (and consequently no. of cells needed)
depends on no. of lobes.
- Invert plates and incubate O/N.
Prepare antibody plates: (200ug/ml ab - 2mls medium).
Note: You can add antibody on reconstitution day but not really necessary.
Place reconstituted lobes on filter discs corresponding
to antibody treatment:
- No treatment (from start of experiment)
- Rat Ig control
- No antibody control
Need 8-10 lobes minimum per test.
Every 6 days: Change
medium (+/- antibody).
Every 2 days: Wash
over lobes (gently).