I have tried isolating leukocytes from whole blood by Histopaque (sigma), but when I checked it in microscope, I found lots of platelets along with leukocytes, I tried washing, but it din't help much.
Hi,
you should be able to isolate them. Although the method is relatively simple there are a few critical steps. Use the Histopaque as specified and make the gradient from H119 and H1077. Optimise how much of the sample you place on your gradient. Either dilute cells or use more gradients with less sample volume. How much sample is used is critical as too much will overload your gradient and you will not get the required separation. Also, when you are overlaying the two different densities and the sample, do it very slowly so that you do not get mixing of the gradient. One should really just float on top of the other. When you are carrying the gradient from one place to another, walk slowly and steadily so that the gradient does not mix and allow a bit of time for the gradient to settle before loading the sample. Also, ensure that the recommended speed is used for your centrifuge. It will work!
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods