I am doing this cell sorting experiment, which lasts around 1 hour and a half. The cells are resuspended in FACS buffer (PBS -/-, 0,1% BSA, 20 mM HEPES) and kept on ice until sorting.
After sorting, I see that I have significative amount of death cells. At first I thought it might be the stress of sorting, but in my unsorted control (also placed on FACS buffer and kept on ice) I also get a similar amount of death cells, so I think is that the cells just don't like the buffer/cold.
I was wondering if anyone knew if I can put glucose or something else to make the cells a bit happier while in FACS buffer. I can not use FBS though, cause it is an unwanted stimulus for my cells.
Thanks a lot!
Ariel
Maybe keeping your cells in culture medium would help, if your FACS buffer is not strictly mandatory
Use serum free medium in ice until you bring sorted cells back to tissue culture hood. Then use complete medium for culturing.
You can use PBS containing Ca/Mg. It may probably prevent cell death or you can also use culture medium without FBS.
Hi, here are a couple of things that might help:
1. Coat the collection tubes with 0.1%BSA - This is really IMPORTANT to prevent cells from sticking to the walls and dying (add the BSA to the tubes making sure that all the walls "see it" and then remove it. If you are working on a cell culture hood and using sterile stuff, you can re-use the BSA solution a couple of times)
2. Collect the cells in cell culture media (not sure if you are using this or PBS)
3. Place a tray with ice around the collection tubes so that cells are also kept "fresh" after the sorting.
Good luck!
Hi,
If your cells have a tendency to clump, try using Ca/Mg++ free PBS. EDTA also helps to prevent the formation of clumps. If your cells are clumping due to cell death, try adding DNAse I and magnesium chloride hexahydrate to your solution.
Also, make sure you are using a good viability dye. If you are using a conventional dye (such as PI, 7-AAD), add it just before running your samples. Other viability dyes need to be added before staining the samples.
If you are sorting your cells for a long period of time, remove the cells at regular intervals and put them back into culture. Keep the collection tubes cold.
Hope this helps.
Kindest regards
Veronica
2-3% FBS significantly decreases cell death. Also you should precoat the tubes with 100%FBS i.e (add 1ml of FBS into the tube and gently shake to make sure it contacts as much surface area as possible). This in our case helped to increase the % yeild and viability.
Thanks all of you for your kind answers!! The thing about sorting the cells in medium is that that would increase the autofluorescence, and one of my FPs is very deemly expressed. I also coat the sorting tubes with my serum free medium, and sort the cells directly into medium. I am using ToPro as viability dye, and the cells look good when being sorted. I liked the idea of doing sets of shorter collections and put them back in culture, I will definetly try that.
At first the 0.1% BSA also striked me as too low, but it is what they recommend at the sorting facility. Nevertheless, I will definetely try and increase the BSA concentration.
Thanks again to all of you!
Best wishes,
Ariel
I would avoid using calcium containing medium as your cells clump more. In my experience, best results are with 2-10%FBS in PBS . But you can add media to the collection tube.
Thanks Simon! The thing is that I cannot use Fbs in my serum free system, that's why I use BSA.
Best
Ariel
There are plenty of good suggestions here. I can add a few more that may help:
1) Avoid temperature changes. This can be difficult if you are sorting cold cells with how they work through the machine and sit in the collection tube for extended periods.
2) Can you use a medium without phenol red as your buffer? I have had luck with XVIVO15 (no serum).
3) If your cells are coming out of cryo before sorting, they can be very sensitive. I would suggest resting before sorting if possible.
Best of luck!
Jeff
I would not fix the cells before sorting because you cannot use them for experiments after that. You can only phenotype them. Most people sort cells so they can use them in experiments afterwards.
Simon gave me another idea about your sorting. If you are activating cells after the sort, it can also be useful to rest them overnight before using them. Of course, this can cause its own set of problems with viability/differentiation (depending on you cell type/species). However, if you are hitting them hard afterwords with something like PMA/ionomycin this will cause the fragile, post-sorted cells to essentially explode.
Ariel,
Indeed, you should use higher BSA levels to keep them alive. What kind of sorter are you using? Might be helpfull to find a sorter which uses hydrodynamic focusing to aligne the cells. In my experience using one cell line which was not able to survive the sort because of a different way of aligning. Hydrodynamic focusing is more gentle to the cells since the effect of the pressure difference is lower. Capturing your cells in culture medium is also advised.
Cheers
- Badges
- Science method