Hi, I am using 2 gradients Percoll protocol to isolate neutrophils from whole blood. I prepare gradients of desired density myself on the day of the experiment from Percoll (Sigma 1644). In my recent trials after centrifugation the blood stays on top of both of the gradients (normally it should be separeted into parts and placed in between the gradients). I assumed the Percoll concentration must have been higher than I had calculated. I've replenished my solution and tried again several times and still got the same result. I think my commercial Percoll Stock solution (which is kept at +4C) might have gone off. Is there any way to verify if the source of my problem is faulty Percoll?
Hello Meftun Doğa Başaran
Before you come to any conclusion regarding the Percoll solution, you should try the protocol given below, if it is not similar.
1. Add 10mL of 75% Percoll into a 50mL conical tube using sterile pipette.
2. Carefully overlay 10mL of 62% Percoll on top of 75% Percoll using sterile pipette.
3. Dilute 10mL of fresh blood 1:1 in 10mL of serum-free RPMI 1640 without antibiotics.
4. Carefully overlay 20mL of diluted blood on top of the 62% Percoll using a sterile pipette in a dropwise manner.
5. Centrifuge the 50mL conical tube at 200 × g for 25 min and 400 × g for 15 min.
6. Using sterile micropipettes first collect PBMCs on top of the 62% Percoll layer.
7. Then collect neutrophils positioned between the 75% and 62% Percoll layers and transfer the cells to a new 50 mL conical tube.
8. Add RPMI 1640 to the tube up to a volume of 50mL. Wash the neutrophils by centrifuging at 300 × g for 5 min.
9. Remove the supernatant completely and resuspend the cell pellet in 1mL of neutrophil buffer.
10. Add 10μL of cell suspension to the hemocytometer and count the cell number under the microscope.
To prepare 62% Percoll gradient solution (50ml): Add 31ml (62%) of Percoll to 5ml (10%) of (10X) PBS and 14ml (28%) of ultra-pure water.
To prepare 75% Percoll gradient solution (50ml): Add 37.5ml (75%) of Percoll to 5ml (10%) of (10X) PBS and 7.5ml (15%) of ultra-pure water.
Some important points to be noted:
1. Percoll should be kept at room temperature (22°C–25°C), otherwise Percoll density changes and the gradient centrifugation success will be compromised.
2. The volume of diluted blood may be adjusted depending on the amount of blood drawn per donor. The amount of diluted blood overlaid should not be greater than 1.5 times the volume of the Percoll gradient. For instance, 15mL of drawn blood (i.e. 30mL of RPMI 1640 diluted blood) can be overlaid on a gradient of 10mL of 75% Percoll and 10mL of 62% Percoll.
Best.
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