7 replies to this topic

[Gradstudent78]

Any tips on washing red blood cells and removing the plasma and buffy coat?

I'm centrifuging them at 1500 x G for 15 minutes and washing with saline.  Having a hard time getting all of the buffy coat. I think I ended up lysing the red blood cells as well. Had a slight conical shaped red film developing at the top of the sample against the side of the test tube (was this lysed cells?).  Thanks, any tips would be appreciated.

Also: blood was collected in EDTA tubes.

Edited by Gradstudent78, 31 July 2010 - 03:52 PM.

[bob1]

I would use a lower RCF for the spin, I use 100 RCF for spinning cultured cells.

[Piersgb]

I'd aggree, spin at a lower speed for longer. I've done some blood preps and I spin at 350g for 20 minutes with the brake set to 0. To help sediment the erythrocytes we add 6% Dextran in a Saline solution warmed to 37*C at a ratio of 2.5ml dextran to 10ml cell pellet. You can then top this solution up with further straight Saline solution. Not sure how essential the Dextran step is though and I guess it depends on what you are actually doing with your cells.

How are you treating your sample beforehand? We collect blood freshly into Sodium Citrate at a ratio of around 1ml Citrate to 10ml of blood and mix gently by inverting the tube.

[Gradstudent78]

Piersgb, on 02 August 2010 - 02:36 AM, said:

I'd aggree, spin at a lower speed for longer. I've done some blood preps and I spin at 350g for 20 minutes with the brake set to 0. To help sediment the erythrocytes we add 6% Dextran in a Saline solution warmed to 37*C at a ratio of 2.5ml dextran to 10ml cell pellet. You can then top this solution up with further straight Saline solution. Not sure how essential the Dextran step is though and I guess it depends on what you are actually doing with your cells.

How are you treating your sample beforehand? We collect blood freshly into Sodium Citrate at a ratio of around 1ml Citrate to 10ml of blood and mix gently by inverting the tube.

Only thing we are doing to the samples beforehand is collecting them in the EDTA tubes (inverting 10 times), pipetting them into test tubes, followed by spinning. I'll try spinning at a lower speed. Any tips on more easily removing the buffy coat?

[newborn]

I use g-force at 500 for 10 min, use a narrow tube 15 ml instead of 50 ml Falcon tube. Then suck around the wall of the tube slowly, you can not take out all white cells but around 80-90%.

Another tip is dissolving your red cells before adding saline after the above step. you just shake the bottom of the tube by your finger.

[Piersgb]

I guess it doesn't matter which Calcium chelator you use (?)

I read somewhere that you can draw the Calcium chelated blood into a syringe with the dextran added and leave for an amount of time to allow the RBCs to settle out. This would leave the plasma on top with the buffy coat inbetween the plasma and RBCs. Then you can attach a bent needle and carefully collect the layers from the syinge that have separated out.

More details were on:

www.malariasite.com/malaria/qbc.htm

I can't seem to get on this today though :S

[Gradstudent78]

Ended up spinning it a little slower, that seemed to help and I think I got a hang of removing the buffy coat. Thanks for the tips.

[DaisyMorgan]

Very Interesting


Daisy x
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