I'm working on MDA MB 231 cell line, which is adherent cell line, I have read a couple of articles that collect cells by trypsinization that some resuspend in complete media and others in PBS, which is best?
and another question,I'm trying to optimize the best dose and dose exposure, hence I'm doing several ( Time exposure) what if I freeze those pellets down until all of them are ready so I can do the essay at once.
Hi Bayan
Regarding your first question ..
we recommend resuspention of your cells in complete media.
Regarding your second question
our recommendation yes you can freeze cell bellets in minus 80 till you collect samples for essay at once.
we recommend this paper for you
Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay
Petra Jackson, Lourdes M. Pedersen, [...], and Håkan Wallin
Additional article information
Abstract
The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R 2 = 0.93 for %DNA in tail (%TDNA) and R 2 = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 57–77% and the day-to-day variation was only 2–12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line