steps my work in the process of cell suspension:
1- after the exit microtube(from freezer -80C) containing liver tissues and PBS buffer, Samples are placed on ice until the ice melted
2- then samples transfer into fresh microtube and Added 200 microliter PBS and We started to cut and homogenized tissues by scissors.
3- after the homogenization, samples are vortexed once and put on ice(Due to the large pieces are deposited).
4- After a few minutes, supernatant is removed with microsampler and transfered to new microtube
5- centrifuging with 1170 rpm,7 minute at 4 C
6- removing supernatant and added 100 microliters PBS and vortexed once
7- again removed the supernatant and added 10 microliter PBS and 100 microliter low melting point agarose
8- finally, after the few times Pipetting, solution loaded on slides and covered by coverslips
Do you confirm the above steps?
Never store samples under frozen condition for comet assay. Cells start getting ruptured and you eventually end up in false positive results. It is better to run the comet assay as soon the sample collection . Once you collect the sample of your interest,place them in PBS and rinse twice to remove blood srains and other extraneous tissue parts. Then gently cope into tiny fragments using surgical blade. Transfer the contents to a glass homogenizer (never use piste and mortar )and gently mince the tissue fragments. You will see the cell suspension and centrifuge at 1000 rpm for 10 min. Collect the supernatant and prepare slides for the assay.
Hi Milad,
Never freeze cells without cryoprotection (glycerol, DMSO, aso..) as transitory formation of ice cristals will break membranes. Without proper step by step or specific cryogenisation process, cells will for sure break at thawing . Anyway, fresh tissue is recomanded in your experiments. Moreover, always add DNAse and/or protease inhibitors (depending on your goal) when thawing snap-frozen tissue .
Hi Milad
PBS is just kind of isotonic buffer, usefull in rinsing and fresh tissue preparation but not protective in cell freezing.
For sure the use of PBS alone on freeze-thawed samples is not good. Better to use fresh liver samples (still with PBS). So I suggest you try first your experiment on fresh liver to check if its better. Maybe you'll also need DNAse inhibtors?
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