Hello,
I am a PhD student in oncology. I am working on a project focused at exploring the brain tumor micro-environment. I want to experiment on conditioned medium from glioma cell line GL261 and the primary cortical glial cells . I have never isolated glial cell , so will much appreciate a workable protocol.
I used to use this protocol. I used DMEM+10% FBS and passaged with trypsin. Other use DNAase/papain. For chamberslides, I think I used poly-D or poly-L-lysine coating followed by gelatin but bare tissue culture treated plastic worked basic astrocytes. You are probably actually isolating glial progenitors that mature into astrocytes in the dish. Also, there are irreversible changes when you expose to serum so defined medias (i.e. with DMEM + G2 or N2) also work and will give in vitro morphology closer to in vivo. Ben Barres paper from Neuron in January 2016 was pretty detailed for immunopanning to get purified populations as the cultures below will be mixed.
Day 1:
1. Put 10 ml DMEM/10% FCS in each 75 cm2 flask. Use one flask for every one or two pups (rat).
2. Dissect cortical hemispheres from P1-P4 pups.
3. Using a 10ml pipette, transfer dissected cortices to a 15 ml tube. (Use more tubes if necessary.) Rinse once with cold CMF.
4. Add 7ml CMF/0.25% Trypsin and strongly pipette up and down a few times with a 5 ml pipette.
5. Incubate tube in 37 water bath for 10 minutes, manually shaking tube every 2-3 minutes.
6. Add 7 ml DMEM/10% FCS to quench trypsin reaction. Pipette up and down.
7. Centrifuge suspension for 4-5 minutes at setting 3 on table top centrifuge.
8. Aspirate supernatant and re-suspend pellet in 2ml (more if needed) DMEM/10% FCS. Transfer to new 15 ml tube.
9. Using a mildly flame-constricted siliconized pipette (or 10ml and 1ml pipette), triturate ~15x. If several tubes were used, combine suspensions. Filter with a nylon cell strainer (only for preparation of astrocytes). For microglia use the entire suspension. Aliquot equal amounts to each flask.
Days 2-7:
1. At 24 hrs, give flasks a little shake and aspirate media. Replace with 10 ml DMEM/10% FCS. Shake again, aspirate, and replace media.
2. To obtain microglia, shake flasks for 2 hr. @ 160 rpm @ 37°C. Collect the media and spin @ setting 4 for 5 min. Then re-suspend microglia in a small volume of media and do a cell count. Dilute the microglia to the appropriate density and plate in 10% FCS/DMEM.
Confluency: Separating Astrocyte and Microglia Cultures
1. Put flasks into a plastic bag. Tape well with masking tape.
2. Dry shake 24-28 hrs at 37°C and at 250 rpm on orbital shaker.
3. Remove flasks from bag. Wash flasks in ethanol, or flame caps.
4. Remove media from flasks. (The media contains the microglia so save for microglial cultures). Wash twice with 5-10ml CMF.
5. Add 5ml CMF/0.25% Trypsin to each flask. Swirl, then incubate 8-10 minutes.
6. Bang flasks to loosen cells. Collect this suspension.
7. To the trypsinized cell suspension, add an equal volume of DMDM/10% FCS.
8. Centrifuge suspension for 4-5 minutes at setting 3 on table top centrifuge.
9. Aspirate supernatant and resuspend pellet in DMEM/10% FCS with mild trituration. Estimate cell number using hemacytometer.
10. Dilute astrocytes in DMDM/10% FCS and plate on polylysinized plates and/or dishes. Use for experiments within 7 days. You may want to switch to serum-free before experiments.
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