I had recently conducted a LDH cytotoxicity assay with my transfected cells as target cells and antigen activated T cells as my effector cells. The transfection efficiency is medium (50-60%) for the plasmid and the cells lose their expression over time and with each passage.
I want to normalize my cytotoxicity data considering the fact that some wells will have more positive cells compared to another.
Can anyone help me with this situation?
Dear Kasturi,
Attached please find a paper "The Impact of Transfection Mediated Toxicity -
Gene Expression and Cytotoxicity Analysis of
Transfection Reagents " that might help you in normalizing the cytotoxicity experiment. I have copied a paragraph which I believe to be important:
LDH Cytotoxicity Assays
Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells. Hela cells were seeded in a 96‐well tissue culture plate at a density of 6 × 103 cells per well and incubated in MEM without Pyruvate, with 10% FBS for 24 hours. Cells were then transfected with the complexes of Luciferase encoding pDNA (pCI luc) and transfection reagents; TransIT‐LT1 (Mirus
Bio), TransIT‐2020 (Mirus Bio), and Lipofectamine 2000 (Life Technologies) according to manufacturers’ protocols for 16‐24 hours. At the end of treatment, the media was collected to measure LDH activity using an LDH‐cytotoxicity detection kit (Roche Diagnostics) according to manufacturer's instructions.
Hoping this will be helpful,
Rafik
Thanks Rafik, I will take a look at this paper and will get back to you if things work out
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