Hello Namritha,

After procuring MCF-7 cells form the supplier, you need to follow a quick thaw process.

The protocol for thawing is as follows.

1) Remove cryovial from storage and hold the cryovial on the surface of the water bath at 37 degree C, and occasionally flick it gently during thawing until only a small frozen piece of ice is still visible. Do not leave the cryovial unattended during the thawing process because thawing takes only a minute or two.

2) Decontaminate by spraying the vial with 70% Ethanol and transfer cryovial to a sterile hood.

All following steps should be performed under sterile conditions.

3) You may add warm complete media drop-by-drop into the cryovial containing the cell suspension, slowly over a 40-second period. The final volume should be around twice the volume of the cell suspension (for instance, add about 1 ml of complete media to a cryovial containing 1 ml of cell suspension). Be careful not to exceed the capacity of the cryovial.

4) Transfer the diluted cell suspension into a 15ml centrifuge tube containing 8ml of warm complete media. For cells preserved in a freezing medium containing DMSO, it is recommended to wash out or dilute the DMSO immediately post-thaw because DMSO harms the cells at higher concentration.

5) Centrifuge the cell suspension at low speed 250 x g for 7 minutes to prevent cell damage.

6) Decant the supernatant and tap the cell pellet.

7) Resuspend the cells in 5ml pre-warmed complete media and transfer the cells into one T-25 cm^2 cell culture flask.

8) Incubate at standard growth conditions overnight (37 degree C at 5% CO2).

9) The next day after the cells have adhered, change the media with fresh complete media.

Yes, passage 3 may be used for MTT assay provided the cells are in good health having all the necessary characteristics namely, normal cell morphology, doubling time (around 28-30 hours), etc., because sometimes some cell lines may require a longer time to recover from stress. At the same time, you need to prepare a frozen stock of MCF-7 cells which you would require for future experiments. As you know that with repeated subculturing the cell lines show genetic drift due selective pressure. Therefore, you need to freeze cells at early passage so that the cells maintain their characteristics whenever you perform any cell-based assay for accurate results.

To prepare frozen stock, you need a large number of cells. For this purpose, you will have to grow MCF-7 cells in larger flasks (T-75 cm^2). The freezing media for MCF-7 cells is 70% medium, 20% FBS, 10% DMSO.

Another important point to make note of is that while performing MTT assay using MCF-7 cells, use complete media without phenol red because phenol red and formazan absorption spectra overlap, and you'll have phenol red absorption added to your formazan absorption readings. If you cannot change DMEM (with phenol red) to DMEM (without phenol red), then still you have a solution to this problem.

HCL can help get rid of the influence of phenol red, and 0.01M is found to be the optimal concentration of HCL that can effectively reduce the influence of phenol red and provide maximum absorbance for MTT assay. Dissolving formazan with DMSO will provide a purple color. But using 10% SDS (w/v) with 0.01M HCL will give a dark yellow color.

One final suggestion, supplement complete media with 2mM L-glutamine.

Wishing you and your colleague GOOD LUCK!

Ensure that the MCF7 cells are sourced from a reputable supplier and have been authenticated to avoid misidentification. MCF7 cells typically require a specific culture medium such as RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. When seeding cells for the MTT assay, ensure that the initial seeding density is appropriate to allow for cell growth during the assay period.

Thank you Malcolm Nobre for your detailed answer and valuable suggestions !

Thank you Christy He for your valuable suggestions!