99 per cent of blood cells are RBC, therefore you could make a simple centrifugation of your whole blood at 1000 g.

You should avoid the coagulation with heparine, sodium citrate, EDTA.

Thanks, i tried actually ,i took samples form myself and put it into 3.2% sodium citrate and then centrifuged it at 200rpm for 10min ie 600g i think...but i was not so successful

We draw human blood into heparinized syringe and follow the below protocol. The citation at the bottom will have the cell wash buffer recipe.

Isolating and Washing Erythrocytes

1) Centrifuge whole blood at 500xg for 10 min at 4 degrees C.

2) Aspirate supernatant (plasma) and add cell wash buffer to erythrocyte pellet. Note: Ensure volume of cell wash buffers double the volume of erythrocyte pellet alone.

3) Centrifuge erythrocytes at 500xg for 10 min at 4 degrees C.

4) Aspirate supernatant and add cell wash buffer to erythrocyte pellet. Note: Ensure volume of cell wash buffers double the volume of erythrocyte pellet alone.

5) Repeat steps 3 and 4 two more times for a total of 3 washes of the erythrocytes.

- This wash procedure is adequate in acquiring a 70-80% hematocrit of rabbit, human, or rat erythrocytes. It has not been tried with mouse erythrocytes.

- If erythrocytes appear to be resuspending, increase the deceleration time of the centrifuge.

- The purity of erythrocyte preparations by this method has been confirmed by Wright staining and flow cytometry.1

- DO NOT store isolated erythrocytes on ice. They are fine at room temperature for 4 to 5 hours. They do need to be used on day of isolation.

1. Hanson MS, Stephenson AH, Bowles EA et al. Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP. Am.J.Physiol Heart Circ.Physiol 2008;295:H786-H793.

@ Madelyn thank u for ur protocol, i will try it, but i have few question: my aim is to surface coat these RBC cells with Biotin and eventually with dye conjugated streptavidin, and for this i will be using NHS-biotin, for which i should avoid TRIS buffer, ur wash buffer contains TRIS, so can i use normal PBS buffer pH 7.4 ?

This probably won't be a problem. However, these cells are much "happier" to have some glucose and protein (BSA) in the buffer. I have seen people wash them in various buffers. It really just depends on your endpoint.

thanks again ,can u please suggest some buffers without TRIS and their composition?

I know that we have used HEPES for RBC experiments but not sure that they were ever washed in HEPES. I will look at papers of Randy Sprague from Saint Louis University as well as Mary Ellsworth from Saint Louis University.

Can I use as an anticoagulant EDTA or citrate instead of heparin? If not, can you explain me why so, please?Thank you!

I do not foresee any problem using other anticoagulants. However, you may need to add additional EDTA or citrate to isolated RBCs to be sure not clots form. I, however, have not done this with any other anticoagulant.

Thank you. When/how can I add additional EDTA/citrate? As the buffer contains CaCl2, should I skip on it?You think heparin is a better anticoagulant?(the trombine is anyway removed after first centrifugation)

I think this will just have to be trial and error. As I said, I have never tried other anticoagulants.

Is ficol method is good for separation of only red blood cells

I have no experience with Ficol gradient and RBCs, but my concern would be that the exposure of RBCs to Ficol may alter the cells.

Thank you for your answer, is there any method to isolate only RBC cells alone.

Hi, what are you going to use the red blood cells for? If you want to isolate RBC from the other cells, you just mix hole blood with EDTA K3 al centrifuge at high RPM. Then, sepatare aspiring the supernactant, taking care of eliminate the white blood cell in the interface of plasma, platelets and WBC. Probably you will lost part of the RBC, but you will get a good cuantity of RBC. I hope it would be useful for you. Good Luck

Please my question is how can I harvest pure white blood cells from whole blood. Thanks.

Hi there,

This question has already been addressed with some really good answers, but I thought I might add a bit of literature. I found this thread while looking for some of the same answers, and it may help researchers even further in the future.

In an excellent article (doi: 10.3791/50166, PMCID: PMC3626231) Evans, B.C., et al. provides an extremely helpful and well-written protocol for an erythrocyte lysis assay. Since the work is freely available to the public, and for the benefit of future scholars, I am reprinting part of the article here without permission.

Note again that this is not my work -I do not take any credit for it. Full reference at bottom. 

-------------- 

2. Preparation of Erythrocytes

NOTE: All procedures must be pre-approved by the appropriate Institutional Review Board (IRB), and venipuncture and blood collection must be performed by a trained phlebotomist in order to minimize the risk to the donor. Standard phlebotomy procedures have been published elsewhere.13 [See original work for reference]

--------------

The article then goes on to describe a spectrophotometric method of evaluating the degree of lysis and how to process the obtained data. 

Article attached, and full reference here: 

Evans BC, Nelson CE, Yu SS, et al. Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs. Journal of Visualized Experiments : JoVE. 2013;(73):50166. doi:10.3791/50166.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626231/

Article Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of...

Edit: 23-Aug-2018: Corrected spelling.

Would anyone have an idea as to wether it is possible to extract RBCs (from fish) that has

a) dried up in an EDTA vacutainer for several months (allthough kept refrigerated)

b) clotted/clumped up in the same sort of vacutainer but for a approx 10 weeks.

I've managed to break everything free by adding heparine.

I am to run these through a Flow Cytometer, but the results are quite far from optimal... I'll have to stain (the nuclei) with PI afterwards, preferrably in intact cells as well. As that's how I've done it with fresher samples

I'm guessing the best chance is to isolate the cells that may still be whole somehow? Problem is that there's so much "else" in the samples and I have no idea at which end to begin.

Hi, 

Though seperating plasma from the whole blood is a very straight forward protocol, I am getting a red colored plasma. Simlar to blood. 

I extract blood from mouse tail vein, hold the samples on dry ice until I am done.  Then process it ( centrifuge at 13200 rpm at 4 degrees) and then store it.

Though I collect the blood in EDTA coated tubes, I get few clots. 

Ehat could go wrong? 

Thanks in Advance.

Hi Madhura,

The problem seems to be that some RBC´s were damaged/lysed during your experiment, which is the reason for the plasma being reddish in colour, as the damaged RBC´s lose their hemoglobin that gets mixed with the plasma. This may have happened if you froze these RBC´s before (may have happened to some while on dry ice?) or due to hard shaking or vortex. Or it could even be that the blood sample is not fully centrifugated and seperated.

Unfortunately I do not have an answer for the clotting. Hope it helps somehow.

Hi.

Thanks Nursakinah and Karya for your answers. I appreciate your help.

It was because of dry ice. Wet ice did not cause any problem later.

Thank you.

Article Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of...

Badri L Aekbote

hi,

I designed a novel peptide that is an inhibitor of acetylcholinestrase.

there is this enzyme on the red blood cell membrane, so I need ghost or free hemoglobin RBC to test my peptide.

we tried this method previously and it works:

Al-Jafari, A. A., Kamal, M. A., Greig, N. H., Alhomida, A. S., & Perry, E. R. (1998). Kinetics of human erythrocyte acetylcholinesterase inhibition by a novel derivative of physostigmine: phenserine. Biochemical and biophysical research communications, 248(1), 180-185.

but nowadays I can't find an ultracentrifuge and I'm struggling to start my thesis.

I try this method too but it doesn't work at all:

Schwoch, Gerhild, and Hermann Passow. "Preparation and properties of human erythrocyte ghosts." Molecular and cellular biochemistry 2.2 (1973): 197-218.

the maximum speed of our centrifuge is 20,000 g.

I'm short of time.

PLEASE HELP ME :((