Klapperich Lab:Culture of HCN-1a cells
From OpenWetWare
Overview
This protocol outlines the steps for culturing HCN-1a cells.
Materials
- T25 culture flasks
- Collagen-GAG solution
- HCN-1a media
- DMEM
- 4mM L-glutamine
- 1.5 g/L sodium bicarbonate
- 4.5 g/L glucose
- 10% Fetal Bovine Serum
- TrypLE Express
- DMSO
Procedure
Basic Aseptic Technique
- Sanitize all work surfaces periodically with 10% bleach followed by 70% ethanol.
- Clean all work surfaces (especially in the biosafety cabinet) with 70% ethanol before use.
- Change or spray gloves with 70% ethanol after they touch any unclean surface.
- Spray gloves with ethanol prior to placing objects in/ removing objects from incubator. Be sure to fully dry sprayed gloves before putting hands in incubator. Ethanol can kill the cells.
- When working with RNA, change gloves at least every 15 minutes.
- Wipe down all objects with 70% ethanol prior to placing in biosafety cabinet or RNA work area.
Starting New Cells
- Coat 4 T25 flasks with collagen-GAG. Pour small amount of fluid into each flask. Leave in BSC overnight with UV light on.
- Make a batch of supplemented DMEM as described above. Adjust pH to 7.35.
- Thaw cells by briefly placing in 37°C water bath.
- Pipette 250 uL of cells into each T25 tissue culture flask. Add 10 mL media to each flask.
- As soon as cells attach (overnight), remove original media. Replace with new media.
Notes
References
Contact
Jessica Kaufman