Purification of buffy coat - Can look through and provide advice? (Apr/06/2008 )

Hi,

I am currently working on a protocol on purification of buffy coat.

It is something like this:

From the buffy coat bag, contents(-60ml) are distributed into 2*50 ml tubes

Tubes are are then filled up with 600 G for 20 minutes, RT.

Upper layer is removed and middle part is collected. RBCs is avoided.

Underlay in Ficoll-Paque and spin at 400G for 20 minutes, RT

PBMC bands are removed and placed in 50ml centrifugation tube.

Tube is filled again with PBS and spin at 600G, 15 minutes and RT (washing step)

wash again for two times

There is an issue about contamination RBCs, so how to get rid of them?

resuspend in 5 ml freezing media (DMEM/F12, 10%DMSO, ?FBS not sure) and count cells

freeze them with Mr Frosty overnight and the next day liquid nitrogen

Well, thats it, can someone look them through and give their opinion?

thanks

-Tkhaw-

QUOTE (Tkhaw @ Apr 7 2008, 08:49 AM)

I am also having problems with contamination of RBCs. I think it is due to the ficoll-paque. I am trying to look for a better isolation reagent than this one. Are you going to culture your PBMC after isolation? If not, maybe you can lyse the RBC by adding BD FACSlyse but when i did this, i find that the viability of PBMC was not good. IF you are not culturing you might want to try it.

-Ellyphant-

Do you see a defined buffy coat layer? I ask because when we do buffy coat preps using bovine blood (ACD anti-coag (10%), 50ml tubes, 20mins @ ~ 3000rpm ( think its ~ 1700g....using a large Sorval Centrifuge) we get a really nice layer of white cells laying right on top of the RBCs. Very careful removal through the plasma layer with a 5ml (thin nozzle end) seropippete allows you to 'hoover' up the white cells with very little RBCs. Takes practise, but you can get a really 'clean' WBC prep. If needs be, after washing layer in PBS and carefully removing supernatant (300g to aid removal of thrombocytes/granulocytes - a real pain in bovine blood preps!) we sometimes lyse the RBCs with a Ammonium Chloride Lysis buffer which we make oursleves - max. 10mins, RT and spin out (now normal 600g washes in PBS). Then a quick underlay with Histopaque (1.083g/ml but you can obviously use the 1.077g/ml if working with human samples) 45mins @ ~ 1200g, RT - we really normally don't have a problem with RBCs using this protocol. We further culture the PBMCs into macrophages, or isolate monocytes and generate mDCs from those. No culture problems observed so far.......

I can send you a more detailed protocol if you like? and recipe for lysis buffer

-celtic_girl-

i did it two weeks ago... sorry for the late reply, i have been hold up by stupid bureaucracy since April in regards to getting the buffy coat in the first place!

Anyway, I separated the bag into two 50ml tubes, roughly 30mls each. I followed the protocol closely and found that the critical part is the underlay. i have to use a pipette aid to underlay it... a pain in the knees literally (have to kneel down while doing it!) Sometimes, using a pipette aid is not really helpful in underlaying, one mistake and you mixed it all up! Fortunately, it worked. I froze them all in aliquots. All i need now is the ammonium chloride lysis buffer, which stated in the protocol given is 1.66% to lyse the RBCs. Also, i incorporated a step with 200G to remove platelets. Seems perfect. Thanks for the offer!

QUOTE (celtic_girl @ Jun 20 2008, 08:02 PM)

-Tkhaw-