Target cells. B-lymphoblastoid cell lines (BLCL) were derived from all subjects

by transformation of PBMC with Epstein-Barr virus. Briefly, 20 106

PBMC were suspended in 10 ml R-20, in the presence of cyclosporine A at a final

concentration of 0.5 g/ml, and 10 ml of supernatant from the marmoset cell line

B95-8 was added. The culture was maintained in 5% CO2 at 37°C for up to 4

weeks to allow transformation to occur. BLCL were cryopreserved in liquid

nitrogen in aliquots of 10 106 cells until needed. For cytotoxicity assays, BLCL

were labeled with 3.7 MBq Na2

51CrO4 (Perkin-Elmer, Wellesley, MA) overnight

at 37°C, washed twice, and suspended at a final concentration of 3 104 to 5

104 cells/ml R-10.

Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes.

Imrie A, Meeks J, Gurary A, Sukhbataar M, Kitsutani P, Effler P, Zhao Z.

J Virol. 2007 Sep;81(18):10081-91. Epub 2007 Jul 11.

 Thank you for your anwsers. 

Alina Radziwon, did you use B cells isolated from donors?

Cyclosporine is a determinant key at growing LCLs as it kills T cells and only B cells stay alive. I used at final concentration of 1ug/ml. also, the initial cells (I used to used PBMCs) number is also a crucial factor. I started with 0.5 millions cells but they did not grow. By far, 3 milions cells work at best to me. The reason behind this because we do not know how many B cells in the collected PBMCs. Samples that have a huge number of B cells are much easier to be transformed into LCL. Another aspect is the microenvironment we provide for the cell growth. As cells tend to physically have contact to each other, plate them out into round bottom 96-well plate, resuspended in 150 ul CSA media and check them every 3 days. This will help them dividing more and quick when you start with under a million cells. Hope this helps.