Do anybody know how to get good mononuclear cell seperation from old blood (not much old, upto 2-3 days that take it for transport). Is there something to get rid off RBCs?
The best way to get red of RBCs is to lyse tem in a solution like this one
http://www.thelabrat.com/protocols/RBCLysisSolution.shtml
but try not leav it longer than 10 -15 minutes on ice ( to ovoid PMN activation) spinning after that at 1000 RMP you wil lsee a visibable whight untarnished plug if you need to you can do an extra incubation after you suspend the plug in the same buffter for an additional 10 minutes on ice then spin again
I have always used 1:4 dilution of the lysis buffer of the whole sample nothing else. you can try on an aliqute of your sample and see if there is a difffrence in yeald when you use the beeds and SIGMA's system.
it is better to use Ficoll-hypaque after RBC lysis to collect Maximal MNCs
my usual protocol is to overlay whole blood diluted in equal amout with pbs on ficall hypaque, and the seperate the monolayer. but if the sample is old, some rbcs also comes in pellet . what is your protocol ? do u first lyse rbcs, then how do you lay it on hypaque. plz clarify?
I usually lyse the cells firste then overlay the pellet on hypaque
i want the lymphocyte protein lysate for western blot. rbcs create problem in actual protein determination. Since i receive samples outside my city, it takes 2-3 days to come to me by courier. so t is haard to seperate monolayer then.
How can i proceed so that i get good mncs with no rbcs, or very little one if they are so difficult to remove.
some people i think first lyse the rbcs wth lysis buffer and then overlay on histopaq, but i dont know this protocol.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods