Hi Shiqiang,

I have not done this myself, but here is parts of the method where they did what you're after:

Memory B-cell staining, sorting and antibody cloning

Staining and single-cell sorting of memory B cells were performed as follows. PBMCs from HIV-1-infected donor N152 were stained with antibody cocktail consisting of anti-CD19–PE–Cy7 (BD Bioscience), IgA–APC (Jackson ImmunoResearch Laboratories Inc.), IgD–FITC (BD Pharmingen) and IgM–PE (Jackson ImmunoResearch Laboratories Inc.) at 4 °C in dark for 30 min. The cells were then washed with 10 ml PBS-BSA buffer and re-suspended in 500 μl PBS-BSA. A total of 66,000 CD19+IgA−IgD−IgM− memory B cells were sorted using a FACSAria III cell sorter (Becton Dickinson) and re-suspended in IMDM medium with 10% FBS containing 100 U ml−1 IL-2, 50 ng ml−1 IL-21 and 1 × 105 ml−1 irradiated 3T3-msCD40L feeder cells42. B cells were seeded into 384-well microtitre plates at a density of 4 cells per well in a final volume of 50 μl. After 13 days of incubation, 40 μl of culture supernatants from each well were collected and screened for neutralization activity using a high-throughput micro-neutralization assay against HIV-1MN.3 and HIV-1Bal.26.

It is from this paper: http://www.nature.com/nature/journal/vnfv/ncurrent/full/nature11544.html

So the main differences are:

Slightly different stimulation: IL2+IL2+feeders

Different plate format: 384 well plate

Sorting: Did not have markers to the BCR (can activate the cells upon sorting)

Time: quite much longer culture time

Hope this helps!

Hi Shiquiang,

growing B cells is a challenging task. I understand that you used frozen memory B cells. I would suggest you use freshly sorted memory B and add IL6 as a supplement. B cells are difficult to clone. You may wish to use at least 10 cells per well if this is compatible with your experiments. TLR9 agonists such as CpG ODN may also be used for memory B-cell expansion, since these require only one signal for stimulation. Dr Lanzavecchia's group in Bellinzona, Switzerland, has extensive experience in human mAb isolation from memory B cells and you should probably contact him for further advice.

Hi Shiqiang.

In my experiment I add a fresh IL-4 medium (the same with yours) (1/2 well volume)every 48H. Moreover, I add the fresh CD40-L at the same period and incubate the culture for 5 days. Regards

Thank you all, Christopher, Mario and Anna.

For Christopher, the paper is intersting because some midifications have been made here. So I will test IL-2 and IL-21 in my experiment too. As for the culture time, I think longer time may be help, but I am doult whether it can also cause the degeneration of mRNA. Anti-IgG also been used for sorting in previous publications and it has been proved to be OK.

For Mario, I do agree that B cells were hard to clone. CpG ODN have been tried in my experiment and showed only a slight increase in IgG level. As for the number of cell seeding in each well, there will raise a risk of matching 10 diffent heavy chain with 10 different light chain for the right one. So usually less that 5 cells have been tried for the purpose. I will write to Dr Lanzavecchia for advice on my experiment. Thanks for the information.

For Anna, thanks for the sharing. But I just do not think that stimulating combination was strong enough to maintain B cell survival. So can I know what is the purpose of your experiment? have any data on proliferation assay been done? Thanks.

Thanks for all the answers. They are indeed very helpful for me.

Best,

Shiqiang

I recommend you to try 3T3-msCD40L Cell Lines as feeder cells.

We used the Human B Cell Culture and Expansion Kit for single B cell culture. It works very well.

http://www.cellron.com/human-b-cell-culture-and-expansion-kit.html