buffy coat count

[moleculargeek21]

I'm working with a fujifilm autogen to isolate DNA and RNA from buffy coat. I'm not entirely sure how much buffy coat to use from my fractionation but the protocol calls for 3 x 10^6 cells. Could anyone give me an idea of how many cells are typically in the average isolated buffy coat and also a good method to dilute this sample to fit my needs. If i'm leaving out any information that you might need to better answer my question don't hesitate to let me know.

Thanks and Cheers

[luisillo]

Depends on the amount of starting material (the blood from where you got the buffy coat). Why don't you try diluting your buffy coats in sterile PBS and counting the cells in an hemacytometer. I don't remember the protocol for cell count in hemacytometer very well, but you can find it quickly in the web.

Good luck

[moleculargeek21]

I'm pulling buffy coat from two purple top vacutainers, between 4 and 5 ml per vacutainer. I do not have access to a hemacytometer.

[luisillo]

I've never worked directly with buffy coat, but using density gradient separation I used to get between 3 and 5 million cells (PBMC) from 5 ml of total blood. Considering this and your protocol, you could use the buffy coat of one tube per assay.

About the dilution, PBS is a good choice. We use it to wash cells which are going to be cultured, so I don't see any problem if you're going to lyse the cells anyway.

[moleculargeek21]

Upon further digging The protocol is using cells from 500 microliters of whole blood. We are using 4 ml of whole blood therefore 8 times the number of cells. Now it is just a matter of converting the quantities to the ammounts needed.

[luisillo]

Good thing you figured out. Keep up the good work!

[moleculargeek21]

My preparation of the buffy coat is as follows.

I receive two purple top vacutainers treated with EDTA with between 2-4 ml of whole blood. I centrifuge these tubes at 2500 rpm for 10 minutes to separate the layers. I carefully pipette the plasma layer and store it at -80. I then fill two 15 ml conical tubes with cold 10 mM tris 1 mM edta and pipette the buffy coat layer with a few of the rbc’s into the vials. This is allowed to sit for 10 minutes on ice and then centrifuged for 10 minutes at 3000 rpm. The supernatant is poured off and another washing is carried out with 10ml of TE, this is centrifuged again for 10 minutes at 3000 rpm. Repeated washings are carried out until a majority of rbc’s are washed away. The pelleted buffy coat is then transferred to a cryo-vial for storage at -80

Would you suggest washing with TE or with a NaCl solution?

thanks and cheers,
-M