Metastatic Model Cell Harvest
A.Barnard 8/31/2013 Teelab
Solutions
1x Clear CMF-PBS for cell harvest
MEGM
Cell Recovery Solution (CRS)
CMF-PBS:Trypsin-EDTA(1:1)
Protocol
Transwell
1. Aspirate MEGM with pipette
2. Wash 2x with 200ul 1x Clear CMF-PBS, remove with pipette
3. Add 62ul of CRS (amount is determined for 24-well plate transwell) and remove into cold 1.5mL tube
4. Add 93ul of CRS again and add into corresponding tube (make sure matrigel has been removed)
5. Let tubes sit on ice for 2 hours in 4C
6. Mix up and down with pipette at 1 hour into incubation
7. After 2 hours, centrifuge @1800rpm for 8-10mins
-Second option is to pool all samples together of same treatment group into 15mL falcon tube
8. Aspirate supernant with pipette
9. Resuspend in 200ul CMF-PBS:Trypsin-EDTA, mix well
10. Incubate for 5 mins in 37C
11. Add 600ul MEGM, mix well
12. Pass through 40um cell strainer (change filter caps if not going through), wash 3x with 400ul
13. Mix well and centrifuge @1800rpm for 8-10mins (Fleet lab) or transfer to 1.5mL tubes and spin
14. Remove supernant with pipette.
15. Resuspend in 200ul 1x clear CMF-PBS or MEGM
16. Freeze in -20C until use
BMCM Media
1. Aspirate BMCM with pipette and transfer to 1.5mL tubes
2. Aliquot an amount to do live vs. dead cell count
3. Freeze samples in -20C
Plate/Well
1. After BMCM has been stored, wash 2x with 300ul 1x clear CMF-PBS, remove with pipette
2. Add 140ul/well of CRS (amount is determined for 24-well plate) and remove into cold 1.5mL tube
3. Add 211.2ul/well of CRS, pipette into corresponding tubes on ice (make sure matrigel has been
removed) and mix
4. Let sit on ice in 4C for 1.5hours
5. Mix up and down with pipette at 45 minutes
6. After 1.5 hours, centrifuge @1800rpm for 8-10mins
-Second option is to pool all samples together of same treatment group into 15mL falcon tube
7. Aspirate supernant with pipette
8. Resuspend in 300ul CMF-PBS:Trypsin-EDTA, mix well
9. Incubate for 5mins in 37C
10. Add 900ul BMGM, mix well
11. Centrifuge @1800rpm for 8-10mins
12. Remove supernant with pipette, resuspend in 100ul 1x clear CMF-PBS or BMGM
13. Freeze in -20C until use
Transwell Membrane
1. Using forceps, move transwell into empty well
2. Wash 2x with 200ul 1x clear CMF-PBS
3. Flip transwell upside down onto lid with forceps
4. Using a sterile needle, cut around the edges of the transwell to detach membrane
5. Use forceps to transfer membrane into empty 24-well plate (can pool same treatments)
6. Add 500ul CMF-PBS:Trypsin-EDTA (or until covers membranes)
7. Incubate in 37C for 10mins, taking out at 5mins shaking gently to agitate cells
8. Collect cells with pipette and transfer to 1.5mL tube
9. Centrifuge @1800rpm for 8-10mins
10. Remove supernant and resuspend cells in 300ul of MEGM or 1x clear CMF-PBS
11. Freeze at -20C