What is the procedure to get the pure erythrocytes from blood sample ?

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Emmanuel Ifeanyi Obeagu

Hi, follow the following prcedure as established already and it will help you immensely.

Obtain 25 ml of blood from an anonymous human donor, drawn directly into K2-EDTA-coated Vacutainer tubes to prevent coagulation.

Centrifuge blood at 500 x g for 5 min, and mark levels of hematocrit (red, lower layer) and plasma (yellowish, upper layer) on tube.

Aspirate plasma gently via a micropipettor, add into bleach, and discard into biohazardous waste.

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.

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.

Split blood evenly into four tubes, correspon

ding 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).

Centrifuge blood tubes at 500 x g for 5 min. Mark levels on tubes, then aspirate supernatant.

Fill each tube to marked line with buffer of appropriate pH (as indicated in 2.6).

Label four 50 ml conical tubes (one per pH to test), and pipet 49 ml of PBS of appropriate pH into each conical tube.

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.

Gregory Barshtein

You can wash the cells at room temperature, provided that the process does not take more than 30 - 45 min.

You can see the protocol in Article Biophysical and Biochemical Markers of Red Blood Cell Fragility

It should be noted that prolonged centrifugation of cells at high levels of "g" is undesirable.

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