According to the previous MTT result, the cytotoxicity of the treatment is quite high. We highly doubt that it might be the result of MTT solution reacting with the nanoparticle solutions themselves. Therefore, we decide to do the LDH and MTT cytotoxicity test of nanoparticles only without cells.
LDH Cytotoxicity Test
1.Add 200ul nanoparticle solutions to each well, 3 wells per concentration. Another 3 wells are added with serum free medium only.
2.Collect 46ul nanoparticle solution to a new well and triplicate each sample.
3.Add reaction mixture to each sample well and incubate at room temperature for 30 minutes in dark.
Blue cap: Catalyst Red cap: Dye solution
4.Measure the absorbance at 495 nm immediately.
MTT Cytotoxicity Test
1. Add 50ul nanoparticle solutions to each well, 6 wells per concentration. Another 6 wells are added with serum free medium only.
2. 50ul MTT-medium solution was added in a concentration of 250ug/ml in dark at 37°C for 3 hours.
3. MTT-medium solution was discarded and replaced by DMSO.
4. The 96 well plate was shake for 15 minutes until solid crystal attached in each well was dissolved.
5. Absorbanced was measured at 540nm and 690nm using BioTek.
Cytotoxicity Test Result
According to the LDH and MTT cytotoxicity test on this day, we found that nanoparticle could react with the test solutions and therefore we could not use these two methods for cytotoxicity. Further test of nanoparticle cytotoxicity should be done later.