Does anyone have a good protocol to make a single cell solution from cultured liver cells for facs sorting?
I confirm Rodrigo's method - but you have to wash the cells with calcium-free buffer (PBS or HBSS) before using the trypsin. Also, if you want to make saving, you can lower the amount of trypsin down to 1ml per T75 and tilting it to make sure you have a thin layer of trypsin everywhere.
Check that most cells have rounded up before adding the full medium to stop the trypsin reaction.
I confirm Rodrigo's method - but you have to wash the cells with calcium-free buffer (PBS or HBSS) before using the trypsin. Also, if you want to make saving, you can lower the amount of trypsin down to 1ml per T75 and tilting it to make sure you have a thin layer of trypsin everywhere.
Check that most cells have rounded up before adding the full medium to stop the trypsin reaction.
Yes, calcium-free buffer (PBS-HBSS) is very important otherwise you would hardly get any cells suspension. I do not have the previous experience about your mentioned cell type but the technique should work for adherent cell culture.
http://www.hiv.lanl.gov/content/nab-reference-strains/html/Protocol-for-Trypsin-EDTA-Treatment-for-Disruption-of-Cell-Monolayers-December-2011.pdf
http://www.hiv.lanl.gov/content/nab-reference-strains/html/Protocol-for-Trypsin-EDTA-Treatment-for-Disruption-of-Cell-Monolayers-December-2011.pdf
Also: don't forget to add an appropriate buffer (e.g. hepes), if the FACS machine don't have CO2 atmosphere (which I doubt...)
If you are having troubles breaking apart HepG2 cells, do yourself a favor and switch your growth medium. HepG2 cells tend to aggregate and pile in many type of medium (e.g. DMEM), but behave very well ( confluent monolayers, no aggregation) if you grow them in Hams F12 medium (with 5-10% FBS). Trypsinization methods used should no longer be a issue.
Yes you can use 0.05% trypsin +0.02_0.05% EDTA for 5-7min it's better to warm the medium to 37C
Trypsin-EDTA solution of 0.25% Trypsin and 0.02% EDTA in Dulbecco's PBS is suitable for single cell suspension. Follow the method accordingly as I follow this method in my lab:
1. Decant the media
2. Wash with 1 ml 1X PBS in T-25 flask or with 500 μl of Trypsin-EDTA solution.
3.Pour-off the 1X PBS or Trypsin-EDTA solution with pipette.
4. Add 500-800 μl of Trypsin-EDTA solution and left it for 4-8 min in CO2 incubator at 37°C.
5. Meanwhile observe the cell under inverted microscope and when the cells get converted into round shape, put the 1-2 ml complete media in the flask and pipetting the cells suspension so that it get converted into single cells.
Note: Sometimes cells are tightly attached with bottom of culture flask, and after putting the Trypsin-EDTA solution in flask, cells are not detached even after 10 min incubation, then Decant the Trypsin-EDTA solution and add 1-2 ml complete media in the flask and pipetting the cells suspension so that it get converted into single cells.
sure. see this link for instance: http://www.dsmz.de/catalogues/details/culture/ACC-180.html?tx_dsmzresources_pi5%5BreturnPid%5D=192
In case the link does not work, go to www.dsmz.de, product catalogue, cell lines and enter the name of the cell line. You will be led to the recipe for subculturing
Yes off course. You can use pre-warmed 0.5 % trypsin and 0.2% EDTA solution in DPBS for approximately 7 - 10 minutes for better result. I suggested you for using pre-warmed trypsin EDTA solution for establishing the optimum temperature so that you can minimize the cell death.
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