I did a flux cytometry and i'm sure that my cells are PBMC (from cow) , but i don't understand why after the centrifugation i have a lot of cells stick to each other (cannot be dissolved in the media).
Thank you!
Dear Ralph!
You read this answer, please:
Peter Jesse Gaskill added an answer
August 1, 2013
I think that many of the above comments address most of the possible problems, but we have some slightly different protocols and when we have had problems in the past is is because we deviated from these.
When we isolate monocytes from whole blood, we dilute it between 1:3 and 1:4 with either RPMI or 1X HBSS, depending on the final application of the cells. After the dilution, we overlay on Ficoll and spin 30 minutes at 23C. Temperature wise, we generally keep the blood and the Ficoll at room temperature, around 23C. If the Ficoll is too cold or too hot the isolation will not work right. Other than that, all our subsequent spins are done at 4C.
Another possible problem is the amount of Ficoll used. The recommended amount is 1 mL Ficoll per 1 mL blood, but people often use less Ficoll. However, we have found that in certain applications (notably flow cytometry) use of less Ficoll results in a lot of RBC contamination.
We also make our own RBC lysis buffer, which we use aand my experiences with commercial RBC lysis reagents have not been great. To make a 10X stock of our RBC lysing solution, dilute NH4Cl (80.2 g), NaHCO3 (8.4 g), and EDTA disodium (3.7 g) in 1L of dH2O. Dilute the 10X stock in water to get to 1X.
When isolating PBMC, it is very important to adhere to the protocol conditions. If the centrifugation temperature and the volume of ficoll deviate from the protocol value, PBMC can be contaminated with other blood cells, erythrocytes, platelets, which can stick together leukocytes.
Hello Alexandr,
Thank you very much for your answer,
I think i did respect all these conditions but i did not lyse my RBC
Maybe the problem is here
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