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Can You Do MTT Assay for Suspension Cells: Answer & Protocol

  • Apr 3
  • 3 min read
MTT Assay protocol for Suspension cells

Can you perform an MTT assay on suspension cells? Yes, absolutely.

However, the standard protocol used for adherent cells—where you simply dump out the media before adding a solvent—will cause you to lose your cells and ruin your experiment. Because suspension cells do not attach to the culture plate, you cannot aspirate the medium without specific modifications to the procedure.

This guide details the specific adjustments required for suspension lines (like leukemia cells, hybridomas, or lymphoblasts) to ensure reproducible, accurate data.



The Core Challenge: Adherent vs. Suspension

In a standard MTT assay for adherent cells, the formazan crystals formed by viable cells settle at the bottom, sticking to the monolayer. You can aspirate the liquid MTT solution and replace it with a solvent (like DMSO) easily.

For suspension cells, the cells—and the crystals inside them—are floating. If you aspirate the media, you aspirate the data. To solve this, you must use one of two methods:

  1. The Centrifugation Method: Spinning the plate to pellet cells before media removal.

  2. The Direct Solubilization Method: Adding a detergent-based solvent directly to the wells without removing the media.


Method 1: The Centrifugation Protocol (Standard)

This method allows you to use standard DMSO or Ethanol for solubilization but requires careful handling to avoid cell loss during the wash steps.


Step 1: Optimization and Seeding

  • Cell Density: Suspension cells often require higher seeding densities than adherent cells to generate a sufficient signal. A range of 1 x 10^5 to 1 x 10^6 cells/mL is common, but you should perform a standard curve first.

  • Plate Choice: While flat-bottom plates are standard for optical density (OD) reading, using U-bottom or V-bottom plates can make pelleting the cells easier during the centrifugation step, though flat-bottoms are preferred for the final read.


Step 2: MTT Incubation

  1. Add the MTT reagent to your wells (typically a final concentration of 0.5 mg/mL).

  2. Incubate for 2–4 hours at 37 C.

  3. Check for Crystals: Under a microscope, you should see purple formazan crystals inside the floating cells or sticking to the cell surface.


Step 3: Centrifugation (The Critical Step)

This is where the protocol diverges from adherent cells:

  1. Transfer the culture plate to a centrifuge compatible with microplates.

  2. Spin the plate at approximately 1000 rpm (or roughly 400 x g) for 5–10 minutes.

  3. This forms a tight pellet of cells and crystals at the bottom of the well.


Step 4: Media Removal and Solubilization

  1. Carefully aspirate or pipette off the supernatant (culture media + MTT) without disturbing the pellet. Do not invert the plate.

  2. Resuspend the pellet in the Solubilization Solution (typically DMSO or Acidified Isopropanol) equal to the original culture volume.

  3. Measure absorbance at OD=570 nm (often with a reference filter at 630-650 nm).


Method 2: The Direct Solubilization Protocol (Recommended)

To minimize error and avoid the risk of sucking up cells, many researchers and commercial kits (like those from R&D Systems or Abcam) utilize a detergent-based solvent (SDS-HCl or Triton X-100) that is added directly to the well. No spinning or aspiration is required.


Step-by-Step

  1. Culture & Treat: Seed cells and treat with experimental compounds for the desired time.

  2. Add MTT: Add 10 µL of MTT Reagent to each 100 µL of culture volume.

  3. Incubate: Incubate for 2–4 hours until purple precipitate is visible.

  4. Add Detergent: Add 100 µL of Detergent Reagent (often SDS-based) directly to the well. Do not remove the culture media.

  5. Long Incubation: Leave the plate in the dark at room temperature (or 37 C) for 2–4 hours (or overnight) to allow the detergent to lyse the cells and solubilize the crystals.

  6. Read: Measure absorbance.

Pro Tip: This method has higher background absorbance because the phenol red and media proteins remain in the well. Always run a "Media Only" blank and a "Cells + MTT + Killed" control to subtract background noise.

Troubleshooting Suspension MTT Assays

Problem

Cause

Solution

Low Absorbance Values

Loss of cells during aspiration.

Switch to Method 2 (Detergent) or increase centrifugation speed/time.


High Background

Media remaining in the well (Phenol Red).

Use a reference wavelength (630-650 nm) to correct for nonspecific background.


Incomplete Solubilization

Crystals are tightly packed in the pellet.

If using Method 1, pipette up and down vigorously after adding DMSO to ensure the pellet is fully dissolved.


Variable Data (High Error Bars)

Inconsistent cell numbers per well.

Suspension cells settle in the reservoir during pipetting. Vortex your cell reservoir frequently while seeding the plate.


Summary of Key Modifications

  • Adherent: Dump media ---> Add DMSO.

  • Suspension (Option A): Spin plate ---> Aspirate carefully ---> Add DMSO.

  • Suspension (Option B): Add Detergent directly to media ---> Incubate overnight.






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