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1 Cultured cells
2 Slides
3 Flow cytometry
4 Xenografts

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Cultured cells

Counting cells

hemacytometer

Vi-cell

Passing cells

Freezing cells

Crystal violet: growth inhibition

MTT Assay (Metabolic Activity/ proliferation proxy)

Exponentially growing cells (ex: UMSCC-1) are seeded into 96-well TC plates in standard medium.

Ideally, duplicate plates containing six replicate wells/assay condition are seeded at a density of 1500 cells in 0.1 ml of medium.

Note that the 1,500 cell recommendation is based upon allocating 24h for establishment on the plate and 24h drug Tx; definitely reduce for longer culture durations.
Remember to set up acellular wells for use as blanks.

For drugs dissolved initially dissolved in DMSO, maintain the same DMOS concentration (ex: 0.1%) in all wells, including controls.

Drug addition is best done by medium replacement (although addition of 2x drug is acceptable): It maintains drug pressure during MTT pulse and will allow all steps to be done in one plate (wells reasonably holds 350uL).

Culture cells with drug for the desired duration.

After the treatment period, 11 or 22uL of [5mg/mL] MTT is added to each well for 2 h (see note) at 37°C to allow MTT to form formazan crystals by reacting with metabolically active cells.

Vol. = 11 uL per 100 uL already in well: objective is 0.5 mg/mL final.

[Aspiration may be done here... consider the needs of your experiment. By default, medium is not removed... plan ahead for required volumes for reading]

The cells are killed and formazan crystals are solubilized overnight at RT [per most protocols; SMH uses 37C] in a solution containing 20% of SDS and 50% of N,N-dimethylformamide [full recipe in lab].

Use a volume equivalent to the current volume (ex: 111uL). Note: this solution may solidify if refrigerated. Warm it in a 37C water bath to melt it before use.

The absorbance of each well is measured in a microplate reader at 570 nm [SMH used A600, which is also in the acceptable range]. The percentage cell growth is calculated by comparison of the A570 reading from treated versus control cells. Be sure to break all bubbles in the optical paths!

Time budgeting: D0: seeding (substantial counting; accurate plating), D1: apply Tx, D2 or 3 (24 or 48 hr Tx): 2h MTT/ consistent duration before SDS/DMF addition, D3 or 4: readings

Variants/ Notes:

Plating can be done in larger plates: scale volumes. Transfer to separate assay plates is required. One advantage of the 96-well approach is that sample transfer to separate assay plates for reading is not required.

MTT duration can range from 0.5-6 hours, due to differences in cell densities and metabolic activity between experimental designs. Purple colored granules should be apparent before you do the solubilization step.

Wavelength: A550-600 is appropriate. [Note: some labs @ UW use 540; this is probably for the XTT aqueous variant] The spec's reference should be >650nM (Anywhere in 630-750nM range is acceptable.. use dual wavelength for reference). Blank against the acellular wells @ A570 (set up blanks in the template).

Modified tetrazolium salts simplify the assay (vs. formazan salts like MTT) as they are soluble: see Roche Cell Proliferation Kit II and consider it (price?).

This is more properly described as an NADH/NAPDH succinate-tetrazolium reductase assay, as opposed to a mitochondrial activity assay: most of the activity is cytoplasmic according to Roche.

v0.7, EAA 082207

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