Embedded Files
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 37oC.
- 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 37oC for 4 hours.
- Add 100uL of the SDS-HCl solution (prepared previously) to each well and mix well with pipette.
- Incubate at 37oC for 4-18 hours. Longer incubation times reduce sensitivity of the assay.
- Read the plate at 570nm using a plate reader.
Report abuse