I need to know how to isolate the MNC's from bone marrow. I know how up to a certain point, and I am hoping you can take a look and fill in the blanks.
So I have 50ml's of marrow and am isolating the MNC's from that.
Steps
1.) Dilute the marrow 1:!. So take the 50ml's, put it in two separate 50ml centrifuge tubes with 25ml's of marrow and 25ml's PBS in each tube.
2.) Layer the diluted blood over ficoll. So put ficoll in 4 50ml tubes, then layer the diluted blood on over that?
3.)Centrifuge for 30 minutes at 400g
4.)Remove layer between ficoll and plasma containing MNC's
5.) Then What???
I need to know what happens now? Where step 5 is, what should happen in order to get MNC's? Also, in step 2, how much ficoll should be used in the 4 50ml centrifuge tubes?
continue by:
5.Wash buffy coat with PBS w/o Ca2+, Mg2+, centrifuge 300g, 10min, 18oC
6. Lyse RBC cells by NH4Cl (Becton Dickinson)
7. Incubate in dark, 20 min, centrifuge
8. Wash with PBS w/o Ca2+, Mg2+
9. If you have collected the buffy coat properly the sample will be the fraction of mononuclear cells
Good Luck
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