I want to know the protocol for Isolating RBCs from whole blood collected in EDTA tubes. I am planning on studying the lipidomics of the RBC Membranes therefore need to isolate RBCs in intact form.
A very good article you will be of great use
Evans, B.C., Nelson, C.E., Shann, S.Y., Beavers, K.R., Kim, A.J., Li, H., ... & Duvall, C.L. (2013). Ex vivo blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs. Journal of visualized experiments: JoVE, (73) .doi: 10.3791 / 50166
For the benefit of future scholars I allow you to extract the part that interests you. However, by scientific ethics, it is obligatory to quote the reference mentioned above.
2. Preparation of Erythrocytes
1. Obtain 25 ml of blood from an anonymous human donor, drawn directly into K2-EDTA-coated Vacutainer tubes to prevent coagulation.
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]
1. Centrifuge blood at 500 x g for 5 min, and mark levels of hematocrit (red, lower layer) and plasma (yellowish, upper layer) on tube.
2. Aspirate plasma gently via a micropipettor, add into bleach, and discard into biohazardous waste.
3. Fill hematocrit tube to marked line (original level of plasma) with 150 mM NaCl solution. Cap and invert a few times to gently mix. Centrifuge at 500 x g for 5 min.
4. Repeat step 2.3-2.4 to wash blood cells again. Then aspirate supernatant and replace with PBS at pH 7.4. Invert to mix.
5. Split blood evenly into four tubes, corresponding to each pH that will be tested. Label the tubes according to each pH to be tested (5.6, 6.2, 6.8, 7.4).
6. Centrifuge blood tubes at 500 x g for 5 min. Mark levels on tubes, then aspirate supernatant.
7. Fill each tube to marked line with buffer of appropriate pH (as indicated in 2.6).
8. Label four 50 ml conical tubes (one per pH to test), and pipet 49 ml of PBS of appropriate pH into each conical tube.
9. Add 1 ml of erythrocytes (same pH) into corresponding tube for a 1:50 dilution. Visually inspect the diluted blood, which should be turbid and will settle if left undisturbed. If no pellet forms, cells have lysed.
Abderrahim Benkhaled Thank you for your answer. I actually need a protocol in which I wouldn't need more than 5ml blood. Is it possible?
No tanks Muhammad but you can possibly adapt the protocol to 5ml of blood
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