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3T3 Cytotoxicity Assay Using Neutral Red

Step 1: Seed
Step 2: Dose
Step 3: Test Article Removal and Rinse
Step 4: Addition of Vital Dye
Step 5: Addition of Solvent
Step 6: 96-well Plate Reading


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  1. Seed
  2. Dose
  3. Test Article Removal and Rinse
  4. Addition of Vital Dye
  5. Addition of Solvent
  6. 96-well Plate Reading


Cell Storage
Cell Ampoule
Cell Culture
96-Well Plate Cultures

The cells to be used for the 96-well assays are stored in a liquid nitrogen freezer. When ready for use, the cells are thawed, resuspended, and transferred to a culture flask, where they will grow and multiply until optimal conditions have been achieved.

Trypsin solution is added to the flask to remove the cells. The cells are resuspended in medium, counted, and “seeded” into 96-well plates using a multichannel pipet.


Serial Dilution Series
Dosed 96-Well Plate

The test material is serially diluted to make a range of doses.

The test article dilutions are added to the inner wells containing cells.

Each plate also contains 12 negative, or solvent, control wells which are used to determine % of control viability.

Test Article Removal and Rinse

Test Material Removal
Test Material Rinsing
Rinsate Removal

The test material is removed by decanting.

The plates are rinsed with a buffered saline solution to remove any residual test material.

Addition of Vital Dye

Neutral Red Addition
Neutral Red Uptake

A solution of Neutral Red, a vital dye, is added to the 96-well plate. The plates are incubated at standard culture conditions to allow neutral red uptake by the cells.

Addition of Solvent

Solvent Addition

After the 3 hour neutral red incubation, excess neutral red is decanted and solvent is added to all wells.

The solvent extracts the neutral red dye contained within the cells.

96-well Plate Reading

Optical Density Determination

The 96-well plates are placed on a plate shaker to fully extract the neutral red and evenly distribute the dye in each well.

A spectrometer measures the absorbance of each sample at a specific wavelength.

The absorbance values (optical density) are then used to determine the viability of each well by comparing the optical density of the each test material treated well compared the negative (or solvent ) control wells.