I have a problem in isolation of PBMC. I use Ficoll for isolation.
My ring (PBMC) has RBC contamination and after isolation I have very few live PBMC.
How long had your blood sample been standing before Ficoll?
Some RBC can occur in the layer, depends on source age disease state etc.
It is best to store blood at room temperature for no longer than a day, best less.
I would agree that it is certainly best to isolate the PBMC straight after blood withdrawal. Additionally, have you considered using percoll for your cell separation?
I use fresh blood for isolation and immediatly after dilution with PBS , it added to Ficoll. I will try your suggestion ( Using percoll ).
In my experience the key factor is to do the layering very carefully. Use a 50 ml pipette, tilt the tube so that you get a large surface of Ficoll to work on and let the diluted blood drip as slowly as possible down to the Ficoll. Once you have a centimetere or so of blood you can start to increase the speed of the dispersion, but be patient.
I usually use Lymphoprep(R) which is similar to Ficoll. It is important that this is at room temperature for the separation. If it is cold, the RBC contamination tends to increase. Is this perhaps the same with Ficoll?
How do you measure the amount of live cells after separation? Take care during resuspension of cell pellet not to pipette too harshly. Never use a pipette smaller than 1 ml.
The temperature is very important. All reagents must be at room temperature, especially the Ficoll.