4. Proliferation MTT Assay

Post date: Jul 10, 2012 8:37:16 PM

4.1 Aims

- Quantitatively assess the proliferative potential of cells

4.2 Materials

- Invitrogen Vybrant Kit (Cat. # V13154)

- 96-well plate

- Plate reader

4.3 Methods

4.3.1 Cell Culture

- Plate ~ 10⁶ cells in 10mL of growth medium in each 100 mm Petri dish.

- Record passage number of cells used

- Incubate overnight at 37^oC.

- Apply necessary experimental conditions (drug, radiation, etc)

4.3.2 Reagent Preparation

- Prepare 0.01M HCl solution from the 12M solution in the lab by preparing a 0.1M stock solution first.

o Take 1ml of 12M HCl and dilute in 119ml of ddH2O. Label as 0.1M HCl.

o Take 2ml from 0.1M HCl solution and add to 18ml ddH2O in a falcon tube. Label: 0.01M.

- Prepare a 12mM MTT stock solution by adding 1mL of sterile PBS to one 5mg vial of MTT (A).

o Mix by vortexing until dissolved

- Add 10mL of 0.01M HCl to one tube containing 1g of SDS (B). Mix gently by inversion until dissolved.

4.3.3 Labelling Cells

- Aspirate the media from each plate, wash with cold PBS, trypsanize and re-suspend in 1mL in eppendorf tubes.

- Count cells using auto-counter and determine the cell concentration

- Follow the 96-well plate map that was previously organized (click here for 96-well template)

- Add the required volume to each well

- Incubate for 48-72 hours based on observed cell density under the microscope.

- Remove the medium and replace with 100uL of fresh media OR PBS.

- Add 10uL of 12mM MTT stock solution (prepared previously)

- Include a negative control of 10uL of the MTT stock solution added to 100uL media only.

- Incubate at 37^oC for 4 hours.

- Add 100uL of the SDS-HCl solution (prepared previously) to each well and mix well with pipette.

- Incubate at 37^oC for 4-18 hours. Longer incubation times reduce sensitivity of the assay.

- Read the plate at 570nm using a plate reader.