Procedure and precautions for MTT experiment of suspended cells

Procedure and precautions for MTT experiment of suspended cells

Operation steps:

1. The suspension cells were cultured to the logarithmic growth stage, washed with PBS, centrifuged 300g for 5 minutes. Remove the supernatant and re-suspend the cells to the appropriate concentration (e.g. 1×10^5)

2. Inoculated cells: A single cell suspension containing 10% cells was completely cultured and inoculated into a 96-well plate with 1000-10000 cells per well and 200ul volume per well.

3. Cultured cells: Incubated at 37℃ and 5%CO2 for 16-48 hours, and then observed under an inverted microscope.

4. Color: Take out the 96-well plate and extract the supernatant from each hole. Add 100ul of pre-diluted MTT solution (5mg/ml, i.e. 0.5%MTT) to each well, make the cells fully contact with mtt reagent, and continue to culture for 4 hours, so that the cells can metabolize mtt and form purple crystals;

5. Dissolve the crystals: For suspended cells, it is recommended to skip some steps and centrifuge directly (1000 RPM x10 min), then carefully suck off the supernatant. Then, 100ul of dimethyl sulfoxide (DMSO) was added to each hole, and the crystal was fully dissolved by oscillating at a low speed on a shaking table for 10 minutes.

6. Colorimetry: On the ELISA instrument, select OD570nm (630nm calibration) to measure the light absorption value of each hole.

Thp-1 cells Line (catlog: CD0122)

K-562 cells Line (catlog: CD0115)

Note:

6. Experimental environment: Ensure that the experiment is carried out in a sterile, dust-free, constant temperature (37℃) and constant humidity environment to maintain the optimal growth conditions of cells.

7. The crystal should be fully oscillated when dissolving to ensure complete dissolution.

8. Cell count: When inoculating cells, make sure to count accurately, as the number of cells directly affects the results of the experiment.

9. MTT solution: MTT solution needs to be used on the go to avoid long-term storage resulting in reduced activity.

10. Avoid cross contamination: During the operation, pay attention to replacing the suction head and the tip of the tube to avoid cross contamination between different holes.

11. Centrifugal conditions: During the centrifugation process, ensure that the speed and time are set correctly to avoid damage to cells.

12. DMSO operation: DMSO has certain toxicity, and it is necessary to wear protective glasses and gloves during operation to ensure safety.

8. Interpretation of the results: The change of OD value reflects the activity of the cells, but attention should be paid to excluding other possible interfering factors, such as the color of the medium, bubbles, etc.

9. Experimental records: record all operation and environmental conditions during the experiment in detail for subsequent analysis and review.

After the above steps and precautions are completed, the accuracy and reliability of the experiment can be ensured, so as to obtain accurate cell activity data.