Hello,
I am currently optimising my protocol in terms of isolating leukocytes from a method by just collecting the "buffy coat" after spinning whole blood at 3000g 20mins.
This is the protocol that I did:
Spin whole blood (~4ml) at 3000g 20mins room temp
Snap freeze plasma in 0.5ml aliquots
Collected "buffy layer" using a pastette
Lysed RBCs in 10ml RBC lysis solution, 15mins, ice
Top up volume to 25ml by PBS
Spin 1600RPM 5mins (is this speed to high?)
Further RBC lysis in 5ml, 15mins, ice
Top up volume to 25ml by PBS
Spin 1600RPM 5mins (cell pellet now visible)
Wash cell pellet with 25ml PBS (this step may not be necessary)
Spin 1600RPM 5mins
Resuspended cells with 1ml
Do a cell count
Aliquoted 1x10^6 cells per eppendorf
Spin 6000RPM 4mins ** - (noticed that cells did not pellet very well and was smeared on the sides of the eppendorf which means cells may have lysed)
My question now is are the speeds i have used too high and what speed do you recommend for pelleting cells in eppendorf to avoid loss of cells. These cell pellets are stored in -20C to be used for generating protein lysates and for DNA extraction until needed.
Thanks in advance!
I faced the same problem when isolating PBMC from sheep blood.can anyone suggest me best protocol.
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