B lymphocyte Isolation from blood

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NathF
NathF's picture
B lymphocyte Isolation from blood

Could someone help me? I need a method for B lymphocyte isolation from blood?

Thank you

Li Li
Li Li's picture
You may use magenatic beads

You may use magenatic beads to either positivie selection, or enrichment of B cells from blood, or by flow sorting.
Li Li

samm
samm's picture
You can use ACK lysis to get

You can use ACK lysis to get rid of RBCs, do a Ficoll density gradient (I think its HyPaque 1.077 from Sigma for human blood - check on this), followed by a Miltenyi bead based purification. If you do not use beads, complement mediated depletion using Thy1 can be done before the Ficoll stage to get rid of T cells and enrich for B cells, though the population will not be as pure.

Fujiwara
Fujiwara's picture
Other option than sorting by

Other option than sorting by FACS or magnetic beads could be the use of a column coated with Protein A or G. You can use a anti-B cell antibody (IgG, which binds to the protein A or G) and then run the cell suspension (from Ficoll gradient or ACK lysis, see samm) to select the B cell population. An eluate to break the antibody-cell binding should be used to recover the cells desired.
It won't give a outrageous number of B cells but could be cheaper than flow cytometry, since this require to have a FACS with a sorter installed.

Invitrogen Dyna...
Invitrogen Dynal Tech Support's picture
Hi

Hi

The best method of isolation will be determinded by your intended down-stream application. How pure do you need the cells to be? Should cells be 'untouched' Are you doing molecular or cellular studies?

We have two great kits for B cell isolatin from human samples:

Option 1: Dynabeads CD19 (cat.no 11143D) can be used directly on whole blood samples and the entire isolation only takes 30 min. Cells can then be lysed on the beads and used in molecular studies, or you can use DETACHaBEAD CD19 (cat.no 12506D) to detach the cells from the beads. Isolated cells will be bead and antibody free and in perfect shape for any downstream cellular application. Purity is >97% and yield is >95%.

Option 2:  Dynabeads Untouched Human B cells (11351D). With this kit you will need to isolate PBMC first, e.g. by ficol gradient centrifugation. This kit contains beads with antibodies that remove all celltypes other than the B cells. The purity will be slightly lower than with positive isolation, approximately 95% and the yield is also lower. However, the cells are 'Untouched', which means they have not been in contact with beads or antibody. Isolation time is approximately 60 min.

Both kits are fully scaleable according to your starting sample.

I hope you find this helpful,

Kristina