We need to separate dead cells out from our neuroblastoma cell line, and continue culturing live ones. Sigma website mentions histopaque 1077 reagent can be used for this purpose (also used to separate RBC and WBC from blood). I haven't seen a reference, nor do I understand how histopaque can do that for all cells of the same size.
For our experiment, centrifugal separation of live/dead cells would work the best. Any comments on histopaque on any other centrifugation based method would be deeply appreciated.
Hi Reeteka,
I follow the Sigma instructions with a few minor changes. I have found the for removing dead cells from live cells I centrifuge at 400g/15 min at RT. The volume of histopaque to use is centrifuge tube dependent. 50 ml tube = ~13 ml of histopaque, 15 ml tube = ~3ml histopaque and for very small volumes I use microfuge tube = ~200 ul of histopaque.
I hope this helps. Good Luck!
Hi Reeteka,
We use this method all of the time to eliminate dead cells from our culture. Just follow the Sigma protocol and you should be fine.
Thank you Daniel! Appreciate your response.
Can you help me understand the principle behind this separation- what causes the difference in density in live/dead cells...any ideas?
Thanks again
Reeteka
density=mass/volume so Dead cells (usually via apoptosis) results in shrinking and increased granularity (less cytoplasm) so the dead cells have a greater density than the live cells and end up on the bottom of the tube. The live cells with a density approximately equal to or slightly lighter than the density of Histopaque 1077 remain at the interface.
We use it all the time for seperation of live and dead lymphocytes. Its an easy protocol and works well. Since dead cells chang their morphology, this method can be used to seperate cells from the same cultrue. If you need any tipps and tricks as of handling let me know! Good luck! :)
Hi Daniel & Claudia,
Instructions that Sigma offers, that I've seen, is in context of blood cell separation. Do you guys follow the exact steps for live/dead cell separation: centrifugation at 400g/30 min at RT.
30 mins seem long to me for cells to remain in suspension - these are otherwise adherent cells.
How much volume of histopaque do you start with?
Thanks so much!
Hi Reeteka,
I follow the Sigma instructions with a few minor changes. I have found the for removing dead cells from live cells I centrifuge at 400g/15 min at RT. The volume of histopaque to use is centrifuge tube dependent. 50 ml tube = ~13 ml of histopaque, 15 ml tube = ~3ml histopaque and for very small volumes I use microfuge tube = ~200 ul of histopaque.
I hope this helps. Good Luck!
Daniel J. Medina I have a question about histopaque. As the sigma protocol, add 3 ml of histopaque in 15 ml conical tube and add 3 ml of cell suspension media. Right?
Then, at this time, cell suspension media is added to the bottom of histopaque in conical tube? or just added to the top of histopaque?
Thank you.
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