Dear Marcela Cárdenas-Tueme ,

Greetings!!

I will suggest you to do MTS Assay. I use the Promega's CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS​). This particular assay consists of fewer steps as it can be performed in the 96-well plates with no washing or cell harvesting steps.

Website Link: https://france.promega.com/en/products/cell-health-assays/cell-viability-and-cytotoxicity-assays/celltiter-96-aqueous-one-solution-cell-proliferation-assay-_mts_/?catNum=G3582

All the best!!

Best Regards,

Samrat

What is your protocol for MTT assay?

Dear Samrat Das I'm currently using a kit from Sigma Aldrich which doesn't require that many steps either, I'd love to try a new one but right now it isn't possible, thank you for your suggestion :)

Leila Hosseinzadeh Anvar I'm using the Cell Proliferation Kit I (MTT) from Sigma Aldrich (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/11465007001bul.pdf)

The protocol is pretty simple:

1. Seed cells at desired concentration, in my case 100,00 PBMCs per well in a 96-well plate in 100 uL of cell culture medium.

2. We incubate them 4 hr at 37ºC, 5%CO2

3. Then, we add our palmitate dosis 100 uM, 300 uM and 500 uM, for 6 h, 12 h and 24 h.

4. Afterwards, we add the MTT reagent (10 uL, 0.5 mg/mL) and incubate it for 4 h.

5. Add the solubilization buffer 100 uL per well and incubate it overnight.

Something is happening when I add the MTT, sometimes we get a good signal (it turns blueish/purpleish) but sometimes it doesn't. I don't know if I have to thaw the MTT in 37ºC, I usually just thaw it in my hands and add it, but as I've said before, sometimes it works and sometimes it doesn't.

Thank you!

1. You can change your solubilization buffer and add 200 ul DMSO.

2. The storage period may form a precipitate. so warm it to 37 and thoroughly mixed. you can also filter MTT reagent.

Just check your reagents, if you are using DMSO or any other to solubalize the crystals.