long-term MS5 cocultures using (transduced) AML CD34+ cells
Protocol - Thawing AML cells
Put tube in waterbath.
Pipet cells on NCS, spin 1200 rpm for 5 min.
Resuspend pellet in 10 mL NCS with 500 μL Heparine, MgSO4 and DNase.
Incubate 15 min. at 37°C.
Centrifuge at 1200 rpm for 5 min.
Resuspend pellet.
Protocol - MoFlo sorting
Resuspend pellet in max. 200 μL PBS with 10% medium or FCS.
Stain with (3- 10 μL) CD34 PE Ab for 30 min. at RT.
Wash with PBS, 1200 rpm, 5 min.
Put cells trough filter.
Plate 30- 40 000 cells on MS5 in 12 wells plate (precoated with 0,1 % gelatine, 1 hr at 37°C) in Gartners medium with IL-3, G-CSF, TPO.
Protocol – long-term MS5 cocultures using (transduced) AML CD34+ cells
CD34+ cells were selected by MoFlo sorting. 40 x 103 cells were plated in 12-well plates pre-coated with MS5 stromal cells. Cells were expanded in LTC medium ( a MEM supplemented with heat-inactivated 12.5% FCS, heat-inactivated 12.5% Horse serum (Sigma, Zwijndrecht, The Netherlands), penicillin and streptomycin, 200 mM Glutamine, 57.2 m M b -mercaptoethanol (Sigma) and 1 m M hydrocortisone (Sigma)) supplemented with 20 ng/ml IL-3, G-CSF and TPO. Cultures were kept at 37ºC and 5% CO2. Cultures were demidepopulated weekly for analysis. In cocultures that generated leukemic cobblestones (L-CAs), leukemic cells could be harvested from these cocultures after 5 weeks to initiate 2nd cocultures on new MS5 stroma.
Protocol – isolation of Cobblestones from MS5
-place PBS on cells for 2 min
-aspirate PBS, add 4 ml of Gartner medium in 75
-scrape cells with cellscraper
-collect cells in 10ml tube, wash T75 once with PBS, add to collected cells
-suspend cells by using:
-pink (18 gauge needle) 3 times
-green (21 gauge needle) 3 times
-blue (25 gauge needle) 3 times
-place filter on 50ml tube, pipet resuspended cells through filter
-wash filter with 5 ml PBS
-put cells in new 10 ml tube, spin down
-count cells, count GFP+ cells
-cells are now ready for FACS analysis, secondary CAFC assays, MoFlo sorting, NOD-SCID, anti human CD45 Miltenyi etc
alternative: do not use the sequential needles for suspending cells, but use trypsine:
Take off suspension cells with 5 mL pipet.
Wash 1-2 times with PBS; collect this together with suspension cells. (Count suspension cells)
Add 1 mL Trypsin/EDTA; incubate 2 min. at 37°C.
Take some medium, 1-2 mL per well, and resuspend in the well, be sure that you collect all cells. (Sometimes the MS5 is difficult to get loose, check under the microscope if all hematopoietic cells are collected).
Centrifuge at 1200 rpm for 5 min.
Stain 30 min. at RT with CD45 Ab.
After staining, wash cells with medium, put them over filter tube.
Sort on MoFlo CD45 positive cells.
Collect cells in Gartners- or alpha-MEM medium.
Collect both suspension and adherent cells of the same sample, replate at least ~100 000 cells on MS5 in 12 wells plate.
| Laatst gewijzigd: | 19 november 2012 16:46 |