I am trying to set up a MTT assay using SH-SY5Y. The protocol I am following advises to use 1x104 cells for each well and 24 hours of incubation before proceeding with the MTT assay. However, I have found that the colorimetic changes is not very visible. Also when I look at the microscope they look 40~50 confluent. What cell density would you advise?
Well, for HT-29, CACO-2, MCF-7 and MDA-MB-468 cells, I used 1x104 cells/ml. O count in a Neubauer chamber and calculate the density of the cells for a final volume of 200ul.
Treating the cells at the con fluency of 60-70 % is ok, but you always need to keep in mind that very high OD values are also not acceptable for publications. Also make sure that your cells are healthy and incubate it for your required time before going to take reading.
Hi Priscilla,
You would need about 80% confluence at 24horus. Even if you are completely repeating the experiment, you may need to adjust your cell densities. Double check that the cell densities used in the paper are used for the same size well as yours. Either your growth conditions are not optimal or your cell numbers are very low for the well size. I think it is the latter. I would seed different densities and see how your cells grow. This way you can even do a nice MTT curve and see how sensitive it is and what is the best cell concentration to seed to give you a small error bar. Have about 3 wells per density.
Cheers
Hi, 1. We have used MEM + F12 + 10 % FBS (45: 45:10) medium for culturing SHSY-5Y cells line.
2. For MTT Assay cells were taken from of 60-70 % confluent flask and cell viability count done by trypan blue test (viability was 95%).
3. Cell count adjusted with dilution with medium (5% FBS) final concentration was 2x104 cells/100ul (2x105 cells/ml) were seeded in 96 well plate and incubated for 24hrs in CO2 incubator.
4. After 24hrs treated with different concentrations of test and standard compounds. again plate kept in incubator for for 24hrs.
5. After 24hrs 20ul of MTT solution in PBS (5mg/ml) directly added into plated and incubated plate for 4 hrs.
6. After 4 hrs formazan crystals were observed under microscope. Medium was removed and crystals were dissolved by adding in 50ul of DMSO. O.D. was in medium control was 0.45.
Good luck.
Hi,
its better to keep 80% confluence to carry out MTT assay.dont forget to keep triplicates for each experiments.
Hi Again. I keep trying to optimize my MTT assay but had have a lot of trouble to do so, because I got irreproducible results, every time I run the assay.. I think it may be due to some technical factors such as my pippeting technique. I was thinking that it would be great if you could share some tips about how you prepare your samples for the assay (Do you change tips for every well?, when working with different densities you start making the high density alicuot and then made further dilutions or you part from the mother stock?, Is there any special considerations performing cell counting?, Do you change media before adding MTT?, Have you had problems associated with the edge variability in a 96 well plate? ). Also, could you please provide me and specific absorbance data so I can compare with mine. For example in average I get and absorbance of 0.250 when using 30,000 cells in a 96 well plate. Thank you
Also, do you have approximate numbers of cell counting when cells are confluent in t25, t75 and T225 flasks?
Do you differentiate cells in wells (96 well plate) or in a flask and transfere them prior to the treatment?
you can seed more cells. just be careful to perform MTT before 85% confluence.
if you observe 40-50% confluence you should have less than 1,5 fold increase in cell number (1.5x104). try seeding 2-2,5x104 cells... you should stay under 100% confluence after 24h.
Hello. I know this is quite an old question, but I would like to air my opinion.
For SH-SY5Y cells, if you are using the cells without differentiation them into neuronal-like cells, then a 70% to 80% confluency is optimum for the cells to proceed with MTT assay. However, if you are differentiating them, then it is a bit tricky. As long as the ends or "neurites" are touching each other, it is considered confluent, thus if you see there is a lot of spaces between the cells but the ends are touching, it is considered confluent.
For the first, you could seed and then transfer them to another flask. But for the latter which you had perform differentiation, reseeding might affect the differentiated neuronal morphology, which i would not recommend.
It is always wise to plan ahead and seed the optimum cell density so that after the differentiation process, all the ends of the cells are touching but not packed!
Thank you. Do correct me if I am wrong and if you have better suggestions.
- Badges
- Science method