I have seeded AGS cells (adherent) in a 96 well plate twice for now. There is a doubling time is 22 hours. But even after 24hrs, the distribution of cells in a well is not appropriate. They appear clumped in corners. I have seeded with a density of 10000 cells/well after counting in a haemocytometer.
NISHAT,
Do you have the reference to send to me?
José Walter
I exactly do not know from where the protocol was copied but I am following what was taken from my seniors in the lab. I add 200mic ltrs of complete media (Ham F12) to each well and then calculate cells from T75 flask with the help of Haemocytometer. The cells are averaged by counting all the four corner squares and then the formula I follow is: cell number * dilution factor * 10^4 and by this quantity I calculate and dispense 5000-10000 cells per well. I do everything same for every well. I keep it for 24hrs and check for treatment with my drug. But even after 24hrs of seeding the cells appear in their respective morphology at the corners of wells and the centre of each well looks empty!(
Nishat I'm afraid this is quite a common problem. Cells (for some reason) like the corners of wells and will tend to "gather" there. There are few things that you can try.
a) Titre the cell amount that you add per well (i.e. 1x10e4 - 5x10e4 per well). Obviously the density of the cells is dependent on the cell size and how long you want to keep them sub-confluent in the wells. 1x10e4 is quite low and I expect you to get a more even distribution with a slightly higher density.
b) Alternatively after adding the cells you could put the plate in a centrifuge (if you have one capable of taking a culture plate) and briefly pulse the plate (1 min, low speed). This may help the cells adhere more evenly (rather than letting them "drift" down in the well where,rather like leaves, they gather in the corners. If you pulse them directly onto the well surface they hopefully will stick).
c) pre-coat the wells with e.g. poly-lysine. This may help the cells stick down more quickly.
Good luck and sorry I can't be more help!
Gary
Thank you all of you for the suggestions.
I got an even cell layer today in all my plates. The difference was that I actually mixed the cell pellet of trypsinized AGS cells in the whole media I wanted for plating all the 96wells. And one more thing I did was I kept my plates in the hood itself for settling down for 20min. Moreover, I used a multi-channel pipette this time!
At 12hrs and 24hrs I got 100% cell viability. But after 48hrs of incubation the viability of control(i.e., only cells ,no treatment) is far less than the viability of treated cells. That means according to the rsults the treated cells are 150% viable?? How is this possible?? Kindly help me with this.
Repeat to see if you get the same result.
However where did your 150% comes from? Hows your calculation. What is the "viability" of treated cell at 12h and 24h? Do you know what mtt test for? It doesn't directly test for viable cell if you didn't know that.
yes I know it is based on checking cell metabolic rate with respect to mitochondrial enzymes. But if I consider the OD reading of positive control i.e., only cells, as 100% and calculate other readings it is showing the other ODs as more than 100%. For e.g.: cell control OD is 0.473 then treated OD was much more than this.
I guess one possibility is that the compound you are adding to your cells fluoresces at around the same Ex/Em as the MTT assay. Try adding some to a well with media only and see if it is higher than a media only control.
Another possibility is the substance you are adding is reducing the tetrazolium dye all on its own (independent of the cells). You could check if this is the case by adding substance & MTT into a media only well alongside the above.
@ Gary: I have kept blanks individually i.e., the compound + mgt + dmso. - cells. For 5 mic molar cells I have 5 mic molar blank, similarly for 10, 15 20 etc. So, if there's any chance of reaction or interference of colour then that should be completely eliminated!
moreover theses blanks also give the same reading as the main blank (only media without cells).
Your control can be healthy and you may still get higher level of mitochondria's if your treatment cell grow faster hence more cells.
Does the serum you used with your drugs contain antibiotics? There is chance of bacteria growing in the culture after 48h (but not 12 or 24h)
you should also control the seeding density based on the number of days of experiment. If the wells become highly dense, the cells start to die or lose morphology.
Your compound might caused cell proliferation. Does your compound has any colour pigment? For the uneven or clump in the corner, check that your incubator is leveled. Make sure you mixed well the cells before seeding, the cells will sediment to the bottom after few minutes. Also after seeding onto 96 well plate shake gently horizontal and vertical. Do not swirl the plate. How many technical and biological replicates? What are your test concentrations and do you have any positive control that can kill 50 ot 100% cells? Do you have background control and vehicle control? Your OD of treated need to be normalized to vehicle control (solvent + cells). Compare the solvent + cells with cells only to make sure your vehicle control is not toxic in the first place. It is not that uncommon for low concentration to have higher viability because your cell could possibly have recovery/ survival mechanism kicks thus having more than 100%. Read on hormesis.
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