Hi everyone!
I'm performing a cell lysis to obtain nuclei and I'm having problems of overlysis (cells appear weird shaped and somethimes there is nuclei content leakage).
I'm following the 10x protocol CG000365-RevC that was used before in the lab and by now I diluted the lysis buffer even 80 times... Cells still appear with some leakage.
I saw that there are other protocols for nuclei isolation in 10x (like CG000169-Rev D) in which the lysis buffer does not contain DTT. Could be the reason of the overlysis?
The cells that I'm using are GM12878 and K562.
Any advice is welcome, thank you :')
this depends on the makeup of the lysis buffer. you may want to use a homemade lysis buffer that is less harsh. Sodium deoxycholate specifically punches holes in the nuclear envelope so if that is in your lysis buffer it would be a big problem. there are plenty of cell fractionation methods out there for cytoplasmic lysis and nuclei retention so i would do a literature/methods search
Hello Andrea Rivero Dacruz,
You may use the lysis buffer provided in the protocol given in the link below. Their nuclei isolation protocol has been validated for use on suspension cells like K-562, Jurkat and GM12878. Why don't you try their protocol?
https://www.takarabio.com/learning-centers/automation-systems/icell8-introduction/sample-preparation-protocols/protocol-nuclei-isolation-from-mammalian-cells?srsltid=AfmBOop6g3fJHsE6gzevUkZnHiWSEV2x3oU5UvzAgXN_UpRUL9xiLsGv
Best,
Julie Ann Dougherty Thank you for the answer! We have always used a homemade buffer. I think that we need to adjust the lysis buffer and the wash buffers to our cell lines (less detergent or mild detergent). I didn't know about the detergent that you mention, but I will keep it in mind not to use it for these protocols!
Malcolm Nobre Thank you for your answer! I didn't find this protocol before... For sure I will give it a try :)
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