I would like to isolate lymphocytes from the lung. One of the step requires a density gradient centrifugation step but most protocols make use of pre-made solutions like EasyColl. Has anyone used a percoll gradient instead? What % did you use?
Try this one:
1. Prepare a 54% Percoll solution using 10X isotonic saline (1.5 M) and sterile water such that the final concentration of saline is 0.15M in the entire solution.
2. Prepare a 78% Percoll solution using 10X isotonic saline (1.5 M) and sterile water such that the final concentration of saline is 0.15M in the entire solution.
3. Pipette 3 ml of the 78% Percoll into a 15 ml tube.
4. Carefully and slowly layer 2 ml of the 54% solution on top of the 54% layer (without mixing them)
5. Carefully and slowly layer 5 ml of single cell suspension on top of the 54% Percoll solution without mixing the layers.
6. Centrifuge the gradients at 200 g for 25 min at room temp.
7. The lymphocyte layer will be on top of the 54% Percoll layer and the PMN layer will be at the interface of the 54% and 78% percoll.
This protocol was optimized for human peripheral blood leukocytes. If you are sudying murine cells, you may need to tweak the % Percoll solutions to optimize the yields as the densities of human and murine leukocytes are a little different.
40/70% Percoll gradient enrich lymphocytes from lung filtrate
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