After centrifugation my pillete when dissolved in Alsever's solution /PBS the solution looks like blood again. Isn't it indication of RBC lysis? How to prevent it?
Following the protocol below:
Blood dissolved in Alsever's solution/PBS
Centrifuge at 600-800 g, for 15 mins at 4°C
Discard supernatant. Repeat the steps till supernatant is clear.
A total 7 times centrifugation was done.
When finally pillette is dissolved in Alsever's solution or 0.5% solution prepared with PBS whole solutions turns into blood red.
Can anyone pleas help?
Dear consider the speed of the centrifugation and add normal saline to ensure the integrity of the cells.
Dear Abdullah,
You said you washed the blood cells for 7 times in Alsever's solution. If your cells withstand 7 cycle of washing there wont be any lysis if you finally reconstitute the pellet. If you dissolve the pellet it would be red always. If you want to differentiate how lysed cells look like then add distilled water in blood or cell pellet. Probably your RBC are not lysed and they are intact you can see them under microscope too. And for any reason there is RBC lysis..... then it would occur in earlier washing steps as well. and your supernatant would be bright red. hope it help.
Thank you for your answers.
Dear Gennaro, supernatant is not fully clear or I should say not clear like PBS yet after 5/7 washes. It still has some reddish,yellowish turbidity. And not goes away after 7 washes. What should I do?
I am trying for hemagglutinin assay, once it gave good result but same RBC, same sample later gave fuzzy result.
Gentle mixing of pellet is required. Either gentle tapping or with pipette with wide mouth tip. using conventional tip may also cause cell lysis too.
Please see the images. Plate (1st image) shows results like HA positive virus, though last row was control. RBC from 50 mL centrifuge tube (2nd, 3rd image) used.
4th image from another RBC stock once gave good result though after that test that rbc stock also gave fuzzy result when used again.
Thank you A. M. Gennaro Mam. Alsever's solution component is 8.0 g sodium citrate (C6H5Na3O7•2H2O), 0.55 g citric acid (C6H8O7•H2O), 4.2 g sodium chloride (NaCl), 20.5 g glucose (C6H12O6) for 1 L. It usually needed equal amount of blood to prevent coagulation.
In case of PBS, I think there may be some issues working. Can we use any type PBS saline or any special type only for RBC PBS wash? We currently use 0.01M PBS which contains (2.5 g Na2HPO4•7H2O, 0.22 g NaH2PO4•H2O and 8.5 g NaCl in 1L). This PBS has different composition than that of our regular PBS used in cell culture as well as HA assay (8 g NaCl, 1.15 g Na2HPO4•7H2O, 0.2 g KH2PO4, 0.2g KCl)
Blood samples are fresh daily. Isn't it possible that too many washes and centrifuge for 20 mins may also be affecting.
Alsever's solution is fine. You can use it to wash the cells as well. you can use it for first 2 wash then wash with normal saline or PBS. I believe 4 washes is sufficient. Cut the tip mouth to make it wider if you are re suspending the pellet with tips. Do all the procedure without giving thermal shock. so do not keep blood at 4 degree or refrigeration temperature. good luck.
Is there any difference between rabbit and guinea pig blood sample in terms of RBC total number? I mean how they supposed affect total rbc number? Also any effect of volume of blood?
Dear All, thank for your response. Problems are solved now. Doing the whole process at 25C and trying not to give any heat shock. RBC samples are okay now.
Thanks again.