StemFit® Technical Tips 1
Tips for single-cell cloning via a coating-free method using StemFit® media and iMatrix-511 Recombinant Laminin E8 fragments.
Benefits:
• Superior colony-forming efficiency, enabling efficient single-cell cloning.
• Coating-free protocol: save time with no incubation period or plate coating with iMatrix511.
Protocol: Single-cell Cloning by the Coating-Free Method
2Prepare 10 mL of 10 cell/mL cell suspension by serial dilution with culture medium with 10 μM Y-27632.
3Add iMatrix-511® Recombinant Laminin E8 fragments to the prepared cell suspension and mix thoroughly.
Tip 1 - Correct concentration of iMatrix-511 ECM:
| ECM | Volume (μl) |
Final conc. (μg/ml) |
|---|---|---|
| iMatrix-511 (0.5mg/ml) |
35 | 1.75 |
4Plate 100 μL (= 1 cell) in each well of the 96-well plate immediately.
5Replace medium with fresh culture medium without Y-27632 at least every three days. Around day 8, select single colonies to be passaged to a 24-well plate.
Tip 2 - Extend or shorten culture period between medium-changes for colonies to grow to the appropriate size.
6After washing the colonies with 100 μl of PBS, detach the cells with 50 μl of cell detaching solution (e.g. Accutase/Detachin or TrypLE) and incubate at 37 °C for 10 min.
7Carefully remove cell detaching solution.
Tip 3 - Remove very carefully because the colonies can detach.
8Dissociate colonies by pipetting with 100 μl of culture medium with 10 μM Y-27632. Immediately transfer resuspended cells to ECM-coated 24-well plate with 400 μl of culture medium containing 10 μM Y-27632.
Tip 4 - Detach the colonies one by one as re-attachment can occur soon after adding the culture medium.
9Change the medium to fresh culture medium without Y-27632 at least every three days.
Superior hPSC Adhesion with iMatrix-511 Coating-Free Protocol
Human Embryonic Stem Cells showed equal or superior adhesion in iMatrix-511 Recombinant Laminin E8 fragments and StemFit media using a coating-free, pre-mixed method vs the traditional, pre-coated method. This time-saving protocol enables high adhesion of cells to the culture substrate with no incubation necessary.
Featured Citations
A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells
Nakagawa, M. et al.(2014). Scientific Reports. 4, Article number: 3594.
Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner.
Miyazaki, T. et al. Scientific reports, 2017, 7, 41165