inducing apoptosis in HeLas - UVB irradiation (Mar/07/2007 )

Hi,

I'm trying to induce apoptosis in HeLas and detect apoptosis using a cell viability assay (rezasurin), caspase 3/7 activity assay and dna fragmentation via agarose gel electrophoresis.

I have a handheld UV lamp which has a power output of 12W.

From the various literature i've read, a dosage of 300-500 J/m2 would be sufficient for apoptosis induction. I tried relating the dosage and the power output by some calculations and irradiated the cells for 8s to achieve the required dosage. After irradiation, the cells were incubated for different time points (6hr, 12hr, 24 hr...), and the assays were performed. The cells were still thriving after the incubation and there was no indication that apoptosis have occured.

I have tried irradiating the cells for a longer time (2 min) and the the cell viability was great reduced, however the other 2 tests did not reveal that apoptosis have occured.

Since we don't have a dosimeter, i could not directly measure the dosage and thus wouldn't be able to determine the right dosage to use. I'm afraid that longer irradiation time would kill all the cells, maybe by other ways of cell death; and that an irradiation time too short will not be effective in inducing apoptosis.

I would like to know if anyone uses UVB irradiation for apoptosis induction in HeLas, whom would be kind enough to talk to me.

Thank you.

Hi !

i don't have an answer for you Q. only an observation. I wouldn't have use DNA frag. to determine apoptosis - the referies will most likely turn it down.

you may look on a couple of complementary assays (i recommend at least one form 2 different groups):

1. Viability - MTT/XTT/MTS/Alamar Blue/trypan blue.
2. cell death - LDH release form cells, Hocets dye exclusion/Sub G1 (FACS).
3. Caspase activity - DEVD-x assays, WB for cleaved caspases, PARP cleavage.
4. Annexin-V/PI staining

good luck !