Hi,

I ran into a problem with FACS with the brain tissue with GL26 Glioma:

We implanted GL26 cells into C57bl/6 cells, and extracted the brain tissue when the tumor grows to 4-5mm in diameter.

Here is the dissociation protocol and Percoll protocol:

Liberase or Collagenase/DNase Protocol

1. Place into cell strainer (40um) above 50 mL conical, pour HBSS, smash with small syringe plug. Add HBSS.

2. Centrifuge @ program 2. Dump out liquid.

3. Prep Liberase Mix: add 847 ul HBSS+2%FBS.

4. Add 1-2 mL of Liberase Mix or Collagenase/DNAse(Collagenase (1mg/ml) Type 4 , DNASe (250unites/ml), pipette.

5. Incubate 30' @ 37°C (water bath). Gently mix samples, incubate 30' more for Liberase. 10-20 min for Collagenase/DNAse

6. Add HBSS+2%FBS up to 5 mL, vortex briefly.

Percoll protocol:

1. Centrifuge @ program 2(1250rpm*10min at 4 degree). Dump liquid.

2. Shake Percoll (30% in HBSS WO Ca and Mg), resuspend in 1 mL Percoll. Add Percoll up to 7.5 mL, BARELY cap the lids.

3. Centrifuge @ program 3(1560rpm*25 min at 4 degree). Take caps off gently.

4. Aspirate myelin, then the rest, leave 300-400 ul.

5. Measure % record total volume.

But I did not find any Green cells on the smear slides with the cells or on FACS analysis. Another problem is we do not have the wild type GL26 cells without fluorescence, so we do not have a good negative control for GFP, and the only thing that I can use the Beads without adding any fluorescence...

Could anyone provide me a protocol that works for your study with brain tumor?

Thanks very much for the help in advance! I have tried FACS with several mice and it was frustrating...

Yanrong