Dear Rosita,

I hope that this protocol will be relevant.

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/10771pis.pdf

Thanks, but in this method they did not exactly mentioned that how we can separate all of those three cells!!!!!!!!!!!!!!! 

Hi Rozita Nasiri,

As far as my knowledge, Ficoll histopaque 1077 can be used efficiently for isolation of lymphocytes alone.

you can find the details of the isolation of different WBC fractions in the following article

http://www.ncbi.nlm.nih.gov/pubmed/17284459

After centrifugation  with Ficoll histopaque 1077, platelets will be above the layer (circle) of the lymphocytes. You can collect them by Paster pipet.

You need to have:

1. Ficoll-Hypaque (Histopaque) 1119

2. Ficoll-Hypaque (Histopaque) 1077

3. RBC lysis buffer

4. 1X PBS

5. 15 ml falcon tubes

Set them out at room temperature (RT) for at least 60 minutes:

DiluteFresh blood: 1XPBS 1:1 Blood

Pour 3 mL Hypaque 1119 as the bottom layer

pour 3 mL Hypaque 1077 upper layer , let it sit for 10 min

Slowly & gently add 6mL of 1XPBS 1:1 Blood to create a distinct top layer

Centrifuge tubes at 700xg for 30 min @ RT

Collect the top layer with WBC and a little bit of the bottom layer without disturbing the RBC layer.

Centrifuge at 500xg for 10 minutes

Decant supernatant and resuspend with 2 mL of PBS + 1 RBC lysis buffer.

Incubate for 20 min in RT while rocking gently.

Centrifuge at 500xg for 10 minutes decant supernatant, and what remains is your WBC.