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Gradstudent78 08-02-2010 01:00 AM

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

I'm collecting blood in EDTA tubes, 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.

luisillo 08-03-2010 06:20 PM

Re: Washing Red blood cells
 
I don't know if I got it right: you want to keep red cells and discard white ones and plasma? If this is right why don't you try Dextran sedimentation:

1. Collect blood into a tube containing sodium or potassium EDTA as anticoagulant.
2. Mix 10 parts of EDTA-blood with 1 part of 8% (w/v) Dextran 500 in 0.9% (w/v) NaCl.
3. After 15-40 min at room temperature remove the plasma layer, containing the leucocytes. The time taken for the erythrocytes to settle out depends on the individual blood sample, the initial blood column height and the ambient temperature.

This protocol will also helps if you want the opposite, I mean, if you want white cells instead of red ones.

Hope it helps

Gradstudent78 08-08-2010 02:05 AM

Re: Washing Red blood cells
 
Ended up spinning it slower and I think I got a hang of removing the buffy coat, thanks for the tips.

Zagami 03-01-2011 01:07 PM

Re: Washing Red blood cells
 
Freshly drawn blood have a total specific gravity between 1.052 and 1.064. Considering that blood is constituted by formed elements (erythrocytes, leucocytes and platelets) and liquid medium (plasma), every component a different density show. Red cells have a nominal density about 1.10 g/ml, white cells a density about 1.075, and platelets-plasma a density of about 1.027. Mature human erythrocyte show the highest density, despite has lost nucleus, Golgi apparatus, centrioles, ER and most of its mitochondria, and this because both cytoplasm bulk consist (90-95% of dry weight) of the iron-carrying pigment hemoglobin that is responsible of 80-90 % of high specific gravity (1.092-1.101, measured by means benzyl benzoate/cottonseed oil serial mixture) and, for a small part owed to the aggregation of the erythrocytes among them. Among the sweaters of aggregation can trap leucocytes or platelets, what don't totally allow the obtainment a suspension buffy-coat free. Insofar, is necessary to proceed at several and differential washings-centrifugations, without addition of some gradient medium. As anticoagulant as been used EDTA-K3 and whole blood has been centrifuged for 10 min. at 120 g for obtained platelet-rich plasma (PRP). Discard supernatant and add disodium EDTA buffered saline solution (wash chelating solution [WCS] for minimize erythrocytes aggregation) to reconstitute initial blood volume. Mix accurately and centrifuge at 500 g for 10 min., discard supernatant and add WCS to reconstitute initial blood volume. Mix accurately and centrifuge at 1000 g for 20 min. Drain supernatant and package erythrocytes is buffy-coat free with purity > 98 %.

Zagami 03-01-2011 01:57 PM

Re: Washing Red blood cells
 
my mistake!
change ... to reconstitute per 2 times initial blood volume, instead ... to reconstitute initial blood volume
excuse to me


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