I am isolating HSC (hematopoietic stem cells) from mice and sorting them with standard antibodies in a BDS8 sorter. I also use a viability dye to gate for live cells. Typically we get 5000-6000 HSCs per mouse, so from three mice the number of sorted cells, as shown on the BD software is pretty good. But when I plated those cells immediately and looked under the microscope I did not see any cell. I kept them in culture for 48hrs, still I didn't see any cell. Sometimes, when I get good number of cells, I plate them and again sort them after 48hrs using HSC markers. Again, the number of cells as shown on the software is good but after plating I am not finding any cell. I usually sort at 1-5 flow rate to cause minimal stress to the cells but I am facing the same problem repeatedly. Even with cell lines, technically I am sorting 3000 cells, but in the plate I can hardly find 300 cells. I use media for collecting cells. Looking at the pattern I think the cells are dying after the sort, but I cannot figure out why is this happening? How can I prevent this?
Hello, in my experience I recommend reducing the time the dye is used for cell viability, leaving the samples for less than 5 minutes and observing them under the microscope. I can assure you that this way the cells will be 100% alive. The use of dye for cell viability for many authors is contraindicated overnight. Good luck!
Hello, Rumela Bose
I wonder what the results of staining with live cell dyes are after you have finished cell screening? Be careful that the number of cells displayed by the flow cytometer software may contain debris or dead cells. And HSC is small in size and unclear in morphology, making it difficult to observe directly under a microscope. It is the right approach to use auxiliary live cell dyes or fluorescent markers to verify. You can refer to Adriano Dala Cassanje 's suggestions.
Note:
The most likely reason for the phenomenon you encountered is that the flow cytometer caused cell damage. Even if you sort at a lower flow rate (1-5 times), the pressure, shear force and laser irradiation of the flow cytometer may still damage HSC. HSC is extremely sensitive to the environment, and mechanical stress can easily induce cell apoptosis or inactivation. It is recommended to reduce the flow rate as much as possible, use a low-pressure mode or gentle sorting setting designed specifically for stem cells, and avoid long-term exposure to lasers.
Suggestion:
If the sorting parameters cannot be further improved, you can focus on optimizing your HSC culture conditions. Hematopoietic stem cells have extremely high requirements for the culture environment, and ordinary culture media may not meet their growth needs. Special stem cell culture media should be used, necessary growth factors (such as SCF, TPO, Flt3L, etc.) should be added, and the culture media should be fresh, sterile and at a suitable temperature. In addition, the collection tube should contain a protective agent (such as BSA or serum) to reduce cell adhesion and damage.
And after sorting, transfer the cells to suitable culture conditions as soon as possible to reduce the exposure time of cells in the tube or collection process.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
Lucian Miles I use the cytokines that you mentioned, in my media and I plate the cells immediately after sort (considering it takes 5min to come back to lab from the flow cytometry facility). Also, for normal cell lines it's very strange that the sort count is 3000 but after playing them and waiting for 48hrs I hardly find 300 cells. Does this mean, the rest are all dead by the shock from Facs? Then it's really scary for limited cells like HSC. Moreover before the sort, we lift the cells from culture plate by vigorous pipetting. That may also make the cells weak. We don't use trypsin or TrpLE for HSC. Is there any other way to collect them from the plate without causing mechanical damage and damage to the cell surface markers?
Rumela Bose ,
The situation you describe is very common, especially for fragile cells such as hematopoietic stem cells (HSC), the mechanical and physiological stress during flow cytometry sorting (FACS) and cell collection can indeed cause a lot of cell death.
Cell collection is important, I mention some precautions.
- Before collecting cells, observe the cell status. Adhesion, clumping, aging, etc. will also affect the survival rate of cells after sorting.Avoid using trypsin or TrpLE when collecting cells, especially for HSC, because enzymatic hydrolysis may destroy cell surface proteins. And avoid vigorous blowing.
- When resuspending the collected cells, carrier protein needs to be added: 0.1%-1% BSA or HSA is added to the collection and sorting buffer to reduce nonspecific adsorption of cells to the tube wall and protect the cell membrane.
- Immediately after sorting, the cells are returned to the culture medium. 5 minutes is a bit too long (keep low temperature during transportation).
Lucian Miles yes I always transport the sorted cells in ice. For this experiment where I am using lentiviral transduction, i coat the 96 well plate with retro-nectin and then add the virus and cells. After 48hrs I sort them for mCherry to select for the positively transduced cells. For this I need to collect the cells from the virus plate and do the sort. As I cannot use trypsin, I had to pipette them out. If I do very softly the cells are not getting lifted up and are remaining attached to the base. This is weird as these cells tend to attach very softly on any other plate but for retronectin coated base, they are somehow remaining firmly attached. So, slow pipetting leads to poor collection of cells and while doing fast pipetting, although the number is increased but they are not surviving. I am confused is this the retronectin or the sorter that is harming the cells?
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