Hello dears, we intend to transfect the RAJI cell line. Our attempt using lipofectamine weren't successful. Kindly suggest us a modification of the protocol or alternate protocol to do it?.
Dear Muhsin!
Please You look at the following article:
Article Growth inhibition and apoptosis of human B-cell lymphoma in ...
Cell culture
The human B-cell lymphoma cell line Raji used in this study was maintained in our laboratory.
Transfection with the shRNA expression vector
For transfection, Raji cells in exponential phase of growth were harvested and washed three times with Opti-MEM (Invitrogen, USA) to replace the culture medium and then 5×105 cells were seeded into wells of a 6-well plate and divided into five groups: group 1, normal cultured Raji cells; group 2, Raji cells transfected with pGenesil-1-Bcl-2-1; group 3, Raji cells transfected with pGenesil-1-Bcl-2-2; group 4, Raji cells transfected with negative control plasmid vector pGenesil-1-NC. Transfection was performed according to the manufacturer’s protocols. The ratio of plasmid to Lipofectamine™ 2000 (Invitrogen, USA) was 1:2. Five hours after the transfection, the medium was replaced by the common complete medium again. After 48 h of transfection, cells stably expressing shRNA were established by selection with medium first containing 600 μg/ml G418. The medium was renewed every 3 days. After 15 days selection, the resistant colonies were combined in pools in selective medium. Then, the resistant colonies were further selected by a huge dose G418 (2,000 μg/ml) for 10 days in order to exclude the possibility of non-transfected but G418-resistant colonies, after the huge dose selection of G418, the colonies stably transfected with G418-resistance were amplified and analyzed by RT-PCR, western blot analysis and flow cytometric assays, for subcutaneous tumorigenesis assay in nude mice.
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