I have been sorting NIH 3T3 based on GFP positivity for quite a long time and this has worked fine when all the GFP positive cells are sorted to the same well (of a 24 well or 12 well plate). The problem is: when I try to single sort them in a 96 well plate (1 cell per well) they die (in fact, I never get to see them, so I assume they are dead).
Variables that I already tried and didn't work:
- to sort them into conditional medium
- to sort them into 20% serum medium
I would appreciate very much if you have any suggestions.
I think you lost the single cell during the passaging required from sorting to plating.
I guess that there is a big risk that using whole serum, all the narrow tubing and cell could clog by proteins binding to them, creating a lot of damage
Medium to put the cells in the sorter: 1X PBS by adding HEPES to 25 mM or using HBSS (Hanks's), or cultur medium with out phenolrot,
Medium in the wells: cultur medium or HBSS 20 % FCS or more. So here you can use pure serum. Some medium where are the cells be happy
coating your 96well plate overnight before sort with medium will help. Plus the media used does not buffer well in atmospheric CO2 so using HEPES to make the media buffer well might help too. Single cell sort will always kill a lot of cells. You have to sort at least 2 or 3 96well plates to get few clone coming up...survival is 10% (average) to 50% MAX
There may be something to do with the cells needing other cells around to grow. Not all types of cells can be grown clonally like this. Have you tried sorting small numbers of cells (maybe 10-50) into single wells to see if that works? If that type of experiment worked and you only had problems with the single cell sort, then likely 3t3 cells don't like to be alone.
Another suggestion is that depending on what you will use these cells for afterwards, you may be able to add roc inhibitor to you collection wells, as this helps cell survival after sorting.
Lastly, I assume this is the case, but it wasn't clear in your question, you gate on viable cells first, right?
I was sorting Ramos cells on a FACS Aria and ran into the same problem. Cell lines tend to have much larger cells than PBMC. What helped with recovery and viability was two things:
1. Changing the filter to a larger pore size (This was most critical.) We used a 70uM filter. Because cell lines are larger, they get shredded in typical filters on sorters (they're optimized for PBMC).
2. Collect the cells in warm (37 degree) media and do not put the cells on ice. Once done sorting plate them immediately and place in a 37 degree CO2 incubator.
There was still significant cell death, however, but I was able to recover about 50% of the sorted cells.
I hope this helps.
And dont sort verry high GFP pos cells. Try it with lower green cells
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