I have a problem in GFP based sorting (FACS ) of K4 cells. These cells are subjected to sorting after nucleofection using Cas-9 (and GFP) encoding plasmid. Cells look fine after 24h of nucelofection, splitted and dissolved in 1x PBS or 0,2%EDTA/PBS as sorting buffer. But every time sorted cells (1 cell per well) do not survive. I can only see debris in wells. Any suggestions how can I move around this problem so that they survive after sorting. Thanks
Before sorting into single cells, I suggest you try bulk sorting and see if they can survive. Sorting is a very stressful phenomenon for cells and many cells will die. Troubleshoot the sorting conditions including the nozzle size, speed, and also check if the direction of your sorting is along the center of the well if it goes to the side of the well I experienced a lot of cell death. I hope you will be successful in trouble shotting it.
As Kim said using the 100µm nozzle is recommended for one-cell FACS sorting. Pressure is lower compare with the 70µm nozzle. You can also add FCS (10%) or BSA (0.5%) to your sorting buffer. It help to keep cells alive.
Wish you success.
Use 100 micron nozzle with sheath pressure ~20psi for K4 fibroblast cells. Also, first check with test sort whether test sort is falling in the center of the well or on the wall and make sure to take at least 100 uL of collection media in each well. and Use 1 drop pure or 1 drop single sort mode. Also check the prepared cells for apoptosis using live/dead cell markers. Note: If cells being sorted are hitting the wall of well then there are greater chances that cells will die. Coating the wells with collection media is also mandatory. Also it would be better if you could share the sample facs data. Also please share the details such as Nozzle size, acquisition speed (events/second), sort mode, gating, collection media, so it will be better to understand and help you with this problem.
Hello Khuram Shehzad,
From my personal experience with performing K.I. on hESC (with a Cas-9 expressing plasmid + repair template containing a GFP expression cassette), we used to plate our transfected cells on a large 10 cm culture dish and let them grow until near confluency so that we have enough cells to 1) freeze some 2) perform bulk FACS sorting. The sorted cells were then cultured in 6-well-plate wells. Single clones were obtained by passaging some cells into a culture dish at very low density (and others at normal density, always keep a backup), letting them grow for about one week while monitoring and marking single-cell colonies as they become visible, and finally picking up the clones (with a 200P) before they fuse.
This approach would probably yield fewer clones and take more time than a successful single-cell sorting. But I would suggest to try this safer protocol while optimizing the single-cell FACS method.
Hope this helps!
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