Single cell survival assay
Hanqin Li, Dirk Hockemeyer
Abstract
This protocol describes the experimental procedure used to measure single cell survival rates post nucleofection of human pluripotent stem cells (hPSCs).
General Notes:
- Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Steps
hPSCs are cultured on MEFs as described in the collection "Thawing, Passaging, and Freezing of hPSCs on MEFS" dx.doi.org/10.17504/protocols.io.b4msqu6e
0.5 million hPSCs are nucleofected as described in “Nucleofection of hPSCs" dx.doi.org/10.17504/protocols.io.b4pcqviw
Seed 1/100, 1/500 of cells post-nucleofection into one well of a 6-well plate containing hPSCs medium with Rock inhibitor. Prepare at least 2 wells for each seeding density as replicates.
hPSC medium
| A | B |
|---|---|
| DMEM/F12 | 385 ml |
| Fetal Bovine Serum (FBS) | 75 ml |
| Knockout Serum Replacement | 25 ml |
| L-Glutamine (100X) | 5 ml |
| Penicillin & Streptomycin (100X) | 5 ml |
| MEM Non-Essential Amino Acids (100X) | 5 ml |
| 2-Mercaptoethanol (10,000X) | 50 µl |
| Heat Stable Recombinant Human FGF2 (25 µg/ml)* | 80 µl |
Final volume; 500 mL*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2.
L-Glutamine (100x)
| A | B |
|---|---|
| L-Glutamine, powder | 14.6 g |
| MilliQ H2O | 500 ml |
2-Mercaptoethanol (10,000X)
| A | B |
|---|---|
| 2-Mercaptoethanol | 0.78 ml |
| MilliQ H2O | 9.22 ml |
Heat Stable Recombinant Human FGF2 (25 µg/ml)
| A | B |
|---|---|
| Heat Stable Recombinant Human FGF2 | 500 µg |
| 0.1% BSA | 20 ml |
Final volume: 20 ml
hPSCs Medium + Rock inhibitor
| A | B |
|---|---|
| hPSCs medium | 500 ml |
| Y-27632 (1,000X) | 500 µl |
Final volume: 500 ml
Y-27632 (1,000X)
| A | B |
|---|---|
| Y-27632 | 5 mg |
| DMSO | 1.56 ml |
Change medium at day 3, 6, 8, 10, and then daily until colonies grow to medium size.
Perform Alkaline phosphatase (AP) staining as described in “Pluripotency markers staining” dx.doi.org/10.17504/protocols.io.b4yyqxxw
Image the entire stained well with ChemiDoc (Bio-Rad). Use a white cardboard as background.
Count AP positive colonies, N.
Calculate single cell survival rate by: N x dilution factor/500,000.
E.g. For the wells with seeding density 1/100, the dilution factor is 100.
