red blood cell lysis

[aquilar]

Hi guys.

I have a quick question about blood lysis, and I hope you can give me some pointers.

I am doing a lab technique which requires erythrocyte (red blood cell) lysate.

 

I collect whole blood (using heparin), centrifuge and discard the plasma and white blood cells. The red blood cells are then added to 4X their volume of distilled water and put on ice for 5 minutes ('lysis stage').

 

It is then centrifuged again, yielding this: (sorry the image isn't great quality)

 

 

There are three layers:

- a dark red pellet at the bottom [blue line]

- a cloudy middle layer, very gloopy/gelatinous [pink line]

- a clear pink liquid on top [yellow line]

 

My question is: which of these layers is the 'red blood cell lysate' ? I've been told to use the supernatant, but again I'm not confident which layer this refers to.

 

Clearly it is not the pellet, but is it the liquid at the top, or the cloudy gloopy layer in the middle?

 

Any thoughts would be very welcome! Thanks a million

[Tr0x]

Short answer

The top layer, probably

 

Long answer

The supernatant is generally the top most layer, but it depends on what your desired final product is.

 

My guess is that the "goopy" middle layer contains some clotting factors/platelets as you said this is from whole blood. This would probably not be the supernatant you're interested in if you're going for erythrocyte lysate.

 

It all depends on your centrifugation protocol though, as the layers can mean various things.

[aquilar]

Thanks Tr0x.

 

I think you're right: the top layer is the lysate (cytoplasm etc). The middle layer may be RBC membranes (?) or clotting factors, as you say. I'm using bird blood, so the RBC are nucleated. Maybe the middle layer is DNA.

[ewmon]

I collect whole blood (using heparin), centrifuge and discard the plasma and white blood cells. The red blood cells are then added to 4X their volume of distilled water and put on ice for 5 minutes ('lysis stage').

My guess is that the "goopy" middle layer contains some clotting factors/platelets as you said this is from whole blood.

I think you're right: the top layer is the lysate (cytoplasm etc). The middle layer may be RBC membranes (?) or clotting factors, as you say. I'm using bird blood, so the RBC are nucleated. Maybe the middle layer is DNA.

I've worked with blood which, when centrifuged or allowed to stand, separates into several layers (top to bottom): lipids, plasma, platelets, and RBC's. Aquilar says the plasma and WBC's were removed in the second step. A small amount of RBC's may have been drawn off as well to ensure only RBC's remain. In that case, the platelets were removed with the plasma and WBC's.

 

As for the remaining RBC's, they are heavier than the water. The cell membranes contain lipids and proteins, so they might be in the supernatant, depending on the lipid/protein composition. Almost all of an RBC's lysate is hemoglobin which seems should be heavier than water.

 

The supernatant is clearer than the other three layers, so I'd say that the mixture is probably (top to bottom): water, cell membranes, and hemoglobin. And this jives with the formulation of 80% water (the top layer looks about 80% of the mixture).

 

Either way, the lysate being the hemoglobin, is probably the heavy pellet on the bottom.

Edited August 4, 2011 by ewmon