Reconstitution Method for FTOC

Contents:

 

 

 

Day Before

Prepare DeGuo plates

Medium:

  • 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

 

  1. Take 50 ml of above. Add 0.018g 2-deguo (filtered) (Sigma, Cat. no: D-0901 for 250mg)
  2. Add 2mls per small petri dish.
  3. Control plate has 2ml normal medium.
  4. Place 2x gelfoams (soaked, cut in half).
  5. Place filter disc (dipped in the medium) on each gelfoam.
  6. Place in 37oC, 5% CO2 incubator O/N to ensure sterility.

 

DeGuo Culture

  • Prepare lobes.
  • Make sure they’re fairly clean.
  • Remember: when removing lobes from torso, make sure it is ‘palms up’
  • Place 5-8 lobes/disc.
  • Incubate as before.

Wash every 2-3 days (use pipette with medium from petri dishes and do GENTLY!

 

Reconstitution

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

Terisaki Plates:

  • 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.

Transfer

Place reconstituted lobes on filter discs corresponding to antibody treatment:

  • No treatment (from start of experiment)
  • Rat Ig control
  • No antibody control
  • Tests

Need 8-10 lobes minimum per test.

Post-reconstitution

Every 6 days: Change medium (+/- antibody).

Every 2 days: Wash over lobes (gently).