After 14 days of culture in angiogenic medium, CPCs were detached and migration was induced on TNF-α and VEGF.
Briefly, cells were treated with 0.04 mg/mL SM30 for 2 h in serum-free medium, and after a recovery of 3 and 22 h in complete medium, the cells were detached and centrifuged at 200× g for 5 min.
For flow cytometric analysis, after 4 or 7 days in growth medium, 2×105 cells were detached from the surface of the culture dish using TrypLE select (Gibco), resuspended in PBS containing phycoerythrin-conjugated anti-CD56 (Milteny, clone AF12-7H3) antibodilutedted 1 to 15 for 10 min at 4°C.
After removing the medium adherent cells were detached by subsequent treatment with PBS/EDTA (10 min) and trypsin/EDTA in Ca2+/Mg2+-free PBS (final concentration: 0.05%/0.1%; 5 min) at 37°C and centrifuged.
In short, FR expressing human KB cells grown in folate deficient RPMI medium (Buffalo, NY, USA) were detached by trypsinization and washed twice in ice-cold Hepes-buffered saline solution (pH 7.4) at a density of 1 × 106 cells/mL.
When harvesting cells, floating cells were collected by centrifugation of the aspirated medium, and adherent cells were detached by Accutase™ and collected by centrifugation, these were pooled together in a media volume equal to the original culture volume before cell counts.
At various times after infection of cells with GFP-expressing viruses (VSV, PIV3, and YFV) in 24-well plates, medium was removed and the cells were detached in Accumax Cell Aggregate Dissociation Medium (eBiosciences).
Next, cell medium was aspirated and cells were detached by a 20 minute incubation with cell dissociation buffer (Invitrogen).
At various observation times, the medium was removed and cells were detached from the flasks by trypsin, washed twice with PBS and stained according to the methods specified below.
Seventy-two hours after transfection, cells were detached with culture medium containing 2 units/ml of dispase, cultured in this medium for 24, 48 or 72 h and the percentage of apoptotic cells was quantified as described above.
