isolation of b cells from PBMNCs

[Vanweinberg]

Hi everyone, i have to isolate b cells with facs from PBMCs and i am not sure which antibodies i should use to really get out all b cells (also plasmacells, plasmablast, mature...)

I know that i have to use Cd 45 for WBCs and CD 19 as a general b cell marker but i think i wont get out the later b cells like plasmacells. I use FACS ARIA 3.
Any ideas or experiences?

greetings

[mmorgan]

Originally Posted by Vanweinberg

Hi everyone, i have to isolate b cells with facs from PBMCs and i am not sure which antibodies i should use to really get out all b cells (also plasmacells, plasmablast, mature...)

I know that i have to use Cd 45 for WBCs and CD 19 as a general b cell marker but i think i wont get out the later b cells like plasmacells. I use FACS ARIA 3.
Any ideas or experiences?

greetings

CD45/B220 should get plasma cells. Syndecan-1 is also a plasma cell marker. How come you want everything? Usually people go for a specific subset. You might want to search for memory B cell markers. Also are you going for B1 cells or just B2 ?

[Vanweinberg]

I want to do IGH sequencing, so in fact it would be ok to use PBMCs without additional b cell sorting because the primer for pcr will hopefully bind only to b cells which have a already recombinated heavy chain. Nevertheless my lab chief wants me to do b cell sorting with FACS bevor...So my idea is to get out simply all b cells to make sure that i don't lose any useful cells.
Concerning B1 cells, i think i've read that there are no B1 cells in the peripheral blood?

[CD19]

There are B1 cells in peripheral blood. The older you are the higher B1 cell count you have.

[habdelsa]

I think CD19 is expressed by all B cell stages if am not mistaked.

[CD19]

Originally Posted by habdelsa

I think CD19 is expressed by all B cell stages if am not mistaked.

Yep. From early pro-B cells.