We like to analyze the erythrocytes metabolome and have whole blood samples to prepare. We would like to separate the erythrocytes befor we lyse and extract them. therfore we search a specific erythrocytes cleanup and preparation protocol for LC-MS analyis. So far I haven't found anything uesful.
Dear Christian,
Standard protocol of RBC separation (based on centrifugation 500-750gX 5-10 min ) from whole blood included:
1. Separation of RBC from plasma;
2.Washing with buffer (PBS or HEPES or ....) two-three time.
Thanks, but in this case we will still have other cells like Leukocytes and Trombocytes between the Erythrocytes or not?
I recommend you to remove after each centrifugation upper thin layer of sedimented cells (for leukocytes deplition). If even a small content of leukocytes is critical, you can use fore leucodeplition the standard filters from the blood bank. Ultimately, you can check the contents of RBC suspension by standard methods
I'd do a density gradient (ficoll, polymorphprep,...) to get a nearly pure erythrocyte population. If that's still not good enough, look at immunomagnetic isolation on top of that, e.g. MACS filters.
We use lymphoprep to remove PBMCs followed by use of plasmion for granulocytes. This works well for us
I think that leukodepletion filters from transfusion bags would be good enought, but after you could add the step with immunomagnetic separtion
or maybe if you have flowcytometry with separtion of cells
Thanks..
We will try now centifugation with several wash steps and we have found a way of coagulate the cells with dextran and filter them out.
Washing whole blood one or twenty times does not help eliminating granulocytes. You need a leukodepletion method with a purification factor of at least 1000 (Log 3). Depending on what you're going to do with the samples, you may even need Log 4 leukodepletion filters. A home-made procedure which should provide a good Log3 purificarion, if well applied, is described in the supplementary materal of the following article:
Achilli C, Ciana A, Balduini C, Risso A, Minetti G. Application of gelatin zymography for evaluating low levels of contaminating neutrophils in red blood cell samples. Anal Biochem. 2011;409:296-297. DOI: 10.1016/j.ab.2010.10.019.
And, for the possible nasty consequences of working with RBC samples contaminated by leukocytes (mostly granulocytes), pls look at:
Minetti G, Egée S, Mörsdorf D, Steffen P, Makhro A, Achilli C, Ciana A, Wang J, Bouyer G, Bernhardt I, Wagner C, Thomas S, Bogdanova A, Kaestner L. Red cell investigations: art and artefacts. Blood Rev. 2013;27:91-101. DOI: 10.1016/j.blre.2013.02.002.
If you find the articles of interest, please cite them heavily in your future publications.
Thank you
You may filter the WB to deplete the majority of the leukocytes from the WB first; then use the centrifuge (we used Hettich Zentrifugen RotoFix 32A, 4,000 RPM/RCF, for 5 min.) to spin down the whole blood ( I presumed that the WB collected in EDTA tube). Then remove the top supernatant and the layer (buffy coat) between the supernatant (plasma) and the packed RBC (in the bottom of the tube). The buffy coat layer is full of the thrombocytes and WBCs. There still might be a small percentage of the leukocytes present. Then, you might want to use the PAL filter again to remove the residue microfibrin and leukocytes. You could make a RBC suspension and check under the microscope to see if there is/are any other blood component present after this. Good luck.
Popular andf frequently methods after E. Beutler : "RED CELL METABOLISM" filtered by alppha cellulose or better Whatman No 1 paper filter, but not completly eliminate WBC !
Use density gradient method by Lymphoprep . You will be able to separate erythrocyte very easily.
If your facility has flow cytometry, you might be able to use specific CD markers to stain whole blood sample to be run on the flow cytometry to sort out RBC's.
Yes , cell sorting will be an interesting way to sortout rbc. I'm doing it.
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