Splenocytes were harvested from B6, 1X RBC lysis buffer (eBioscience) was used according to the manufacturer‘s protocol, cell aggregation was easily seen after RBC lysis but their technique support said this phenomenon was something they never experienced before. I wonder if there is any explanation to this condition?
In our lab we pass the cells through a cell strainer after the RBC lysis. It worked for us.
Dear Alexa:
I think that the problem of cell clumping comes from the remaining fatty tissues attached to the spleen after ablation. Fat usually allows the formation of these "cell clumps" when cell suspensions are stored at low temperatures (just a few minutes at the fridge would be enough to see this clumping).
I would suggest you to deeply "clean" the spleen before disgregation and RBC lysis. You can use sharp forceps or scissors to do this.
Good luck!
Alexa,
Another possibility is that DNA was released from damaged cells. Add DNASE to your cells and incubate at 37oC. This may eliminate the clumping.
Alexa,
In addition to the DNAse, you might want to consider adding a collagenase as well.
yes, but don't you first take the spleen and crush it through a cell strainer? This will kill (lyse) some of the cells releasing DNA.
Hi Alexa, we experienced that some times you get allready aggregation during blood collection. There are two things we do. First we use double concentration of EDTA and we sieve the blood through a 20µm pluriStrainer before use. (cell separation or rbc lysis). I hope the will help. Best Michael.
Hi, it's really nice to hear this is not a specific problem to me.
I simply remove it from the culture with careful pipetting. Once if I have smaller experimental setup, I will check it under microscope.
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