I am doing a viability study using leukemia cells and planning on plating them into a 12 well plate.
1. I was wondering how plating efficiency can be calculated for floating cells such as leukemia cells that do not adhere and form colonies?
I found an online calculation that shows
Plating efficiency= Cell Count on D1/ Cell Count on D0*100
Can this be used for determining the plating efficiency for floating cells?
2. Is there a different method used for floating cells during in vitro viability studies that would be equivalent to plating efficiency?
3. My main concern is to be able to factor for that 12 well plate might have different efficiency than a 6 well plate or T25 flask. I want to be able to effectively factor in the efficiency of the 12 well plate into my viability calculations and remove any inconsistencies caused by the plate of choice. What would be a good method to ensure that?
OR Is this not a problem with floating cells as much, as it is for adhering cells which needed a specific surface area and seeding density?
The difference is the floating def, but the well (96,48,24,12,6) just needs to be sufficient for the number of cells that you seed. Just a matter of experience and experimental design you have. We use 2.5 x 105 cells/ml and we put 100 uL per well in 96 well. But this is designed for normal not leukemia lym. so you need to find a good reference to see how many cells should be used.
When I use suspension cell culturing I consider the number of cells per mL. Usually I seed 200.000-400.000 per mL (depending on culturing period, stimulation length, stimulation molecule concentration etc.), and treat at ~800.000 cells/mL. Higher seeding cell concentration might lead to higher doubling rate, reaching the stationary growth phase earlier. Higher cell concentration at stimulation point might alter the cell response. Critical is to use the same treatment conditions (cell no per well per volume, treatment length etc.) in every independent manipulation for the respective assay. Important is to have you cell culture in the Log growth phase. Is up to you to set the correct parameters.
Hi,
for viability studies (MTT assay) in suspension cells with or without treatment (drug, stimulation, etc.) we normaly seed 5.000/200µL in 96well. Adding MTT after 24/48/72h. And it works very well.
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