| Reagent | Amount | Cat# |
|---|---|---|
| MTT | 5mg | 114-056-101CS (Core Store) |
| PBS | 1mL | 298-93-1 (ThermoFisher) |
11 MTT Assay
11.1 Reagents
11.1.1 MTT
Resuspension of MTT should be performed in the hood, with sterile PBS.
One aliquot of MTT solution can be prepared as follows:
Generally, you should resuspend a larger amount of MTT (final volume 20-30mL) and aliquot it out into labelled 1mL aliquots. These can be frozen and stored at \(-20^\circ C\)
11.2 Overview
A typical MTT assay is carried out in a 96-well plate1. The concentrations of drug you use depends on the \(IC_{50}\) of the drug. This may require a bit of optimization. Your lowest condition should always be DMSO only, with a volume of DMSO equal to your highest-DMSO condition2. The volume of DMSO should generally not exceed 0.1%3, and preferably is less than that (0.01% is great). Generally, we do 10-fold dilutions of the drug (ie 10uM, 1uM…)
Tip
With this protocol, if you start on Thursday, you won’t need to come in on the weekend.
11.3 Day 1: Seeding Cells
Note
Prepare a suspension of your cells and count them
| Cell Line | Cells/Well |
|---|---|
| 1A6 | 1000 |
| 5637 | 1000 |
| BV | 1000 |
| HT1197 | 2000 |
| HT1376 | 2000 |
| J82 | 2000 |
| RT112 | 1000 |
| RT4 | 1000 |
| RT4v6 | 1500 |
| SCaBER | 2500 |
| SW780 | 1000 |
| TCCSUP | 2000 |
| UC1 | 1000 |
| UC3 | 500 |
| UC6 | 350 |
| UC10 | 1500 |
| UC14 | 1000 |
| UC15 | 3000 |
| UC16 | 4000 |
| UC17 | 1000 |
| UC18 | 2000 |
Dilute the cells to appropriate concentration for your cell line (see table). Each well will contain \(100\mu L\). You’ll likely want to make one more mL of suspension than strictly needed so you can use a reagent reservoir and multichannel pipette.
Pour cell suspension into a reagent reservoir4, then use a multichannel pipette to dispense \(100\mu L\) of suspension per well.
11.4 Day 2: Adding Drug
- Prepare a serial dilution of your drug. Prepare a DMSO control, which should contain the same volume of DMSO as the highest DMSO, drug-containing condition (this should be your highest drug concentration condition).
Tip
If you only need 1/4 of a 96 well plate for a given drug, you can prepare a serial dilution in 1mL microfuge tubes. Otherwise, use 15mL conicals.
- Aspirate medium from one ‘condition’ (usually 1/4 a 96 well plate)
Tip
Use a filterless \(1000\mu L\) tip on the typical 2mL aspirating tip to help aspirate medium from the wells
Warning
Try not to suck up the cells on the bottom. Tilt the plate towards you and aspirate on the bottom corner to limit then number of cells you aspirate.
- Use a P200 to pipette \(100\mu L\) of drug medium per well.
Tip
You can get away with using two tips per condition, so long as you aren’t using these drugged media for other conditions. Start with the lowest (non-DMSO) drug concentration, then move to higher concentrations with the same tip. Finally, using another tip, add the DMSO medium.
11.5 Day 5: Replenishing Drug
- Repeat Day 2, preparing new drug to replace the old medium.
Note
Depending on the \(t_{1/2}\) of your drug, this may need to replace the drug more frequently. Doxycycline, for instance, needs to be replaced roughly every other day.
11.6 Day 7: Measuring Metabolic Activity
Add a \(1mL\) MTT aliquot to \(11mL\) medium in a 15mL conical.
Aspirate medium from all wells of the 96 well plate.
Add \(100\mu L\) of MTT solution to each well. Incubate for \(1\ hr\) at \(37^\circ C\).
The following steps can be done outside the hood:
- Aspirate MTT solution out of the wells. Add \(50\mu L\) of DMSO to each well. Shake plate for \(30\ sec\) in the SpectraMAX, then read at \(562nm\) and \(660nm\).