The overall goal of this procedure is to prepare anti-cancer metallo drugs and evaluate their cytotoxicity by the MTT assay. This is accomplished by first synthesizing metal-based anti-cancer compounds. The second step is to prepare 96 well plates of cancer cells.
Next, the metal-based anti-cancer compounds are inserted into the cancer cells. The final step is to measure the cell viability by the MTT assay, where the reduction of MTT by mitochondrial and cytosolic enzymes to form zen is measured ultimately the anti-cancer activity and half maximal inhibitory concentration. Or I see 50 values of the compounds are determined.
Generally individuals new to this method may struggle because it's a multi-step procedure that requires the full attention of the laboratory worker and is placed on strict timeline. Visual demonstration of this method is critical as preparation of cell plate is not straightforward because working with viable cells requires following particular guidelines. Working in a glove box, prepare the vanadium five complex by dissolving 0.42 millimole of the ligand precursor in dry THF.
Then add it to a stirring solution of equivalent amounts of vanadium ot tris isop oxide in THF. Stir the reaction mixture at room temperature for 15 minutes before removing the volatiles under vacuum. Next, add a cold hexane and remove it under vacuum.
A dark purple powder should be obtained in a quantitative yield. Weigh eight milligrams of the obtained compound in an epi orph vial. To prepare the titanium four complex dissolve 0.35 millimole of the ligand precursor in dry THF, and add it to a stirring solution of equivalent of amounts of titanium tetra isop oxide in THF.
Stir the reaction mixture at room temperature for two hours. Then remove the volatiles under vacuum to give a yellow product in a quantitative yield. Weigh eight milligrams of the obtained compound in an eph vial to prepare HT 29 cells in a 96 well plate first culture.
The cells in a 75 square centimeter flask as described in the text protocol, remove the medium when the HT 29 cells reach maximal co fluency. After washing the cells in one milliliter of trypsin EDTA solution, add one milliliter of the same solution and incubate for five minutes. Detach the HT 29 cells from the flask and add 10 milliliters of the medium to disable the trypsin activity.
Transfer five milliliters of the cell mixture to a tube. Use a pipette to transfer a few drops of the cell mixture into a counter chamber. The counter chamber includes two parts of five by five squares.
Count the cells in five representative squares of each part using a microscope. Four squares in the corners and one square in the middle. Calculate the estimated amount of cells present.
Sum the cells in the five representative squares of each part. Calculate the average sum of the two parts and divide by 20. Next, divide 0.6 by the quotient.
The received number is the amount of cell mixture required per plate. This calculation refers to a plate that contains 9, 000 cells per well. Using a pipette, combine the amount of the cell mixture required per plate with 13.2 milliliters of the medium.
Add the mixture to a 96 well plate. Using an 11 channel pipette at 200 microliters per well over six lines containing 66 wells in total. One well of each line should remain empty for the blank control.
Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 24 hours to allow the cells to attach to the plate to insert the compounds first, add 200 microliters of dry THF to eight milligrams of the weight compound. Dilute the compound solution further by first adding 60 microliters of THF in nine different einor files. For each solution, use a pipette to transfer 60 microliters from the compound solution to the first einor file.
Then resus suspend and transfer 60 microliters to the next vial and so on until 10 different concentrations are obtained. Proceed to dilute 20 microliters of each concentration in THF with 180 microliters of medium. Also, prepare a solvent only control solution by adding 20 microliters of THF to 180 microliters of medium.
Next, add 10 microliters from each resulting solution to each well. That already contains 200 microliters of the aforementioned solution of cells in the medium to give final concentrations of the compound of up to 200 milligrams per liter. Each line contains cells treated with the compound in 10 different concentrations.
One, control well containing cells treated with solvent only and one well without cells For the blank control, set up the lines to repeat each measurement of the compound three times. Incubate the loaded plate for three days at 37 degrees Celsius in 5%carbon dioxide atmosphere. Prepare the MTT solution as described in the text protocol using an 11 channel pipette.
Add 20 microliters of MTT solution to each well except the blank control wells. Incubate the plate for an additional three hours after incubation. Remove the medium solution from each well and add 200 microliters of isopropanol to each well.
To dissolve the formin, stir the plate for 30 minutes until homogenous. Measure the absorbance at 550 nanometers for 200 microliters of the homogenous solution by a microplate reader, spectrophotometer repeating each measurement on three different days. Next, calculate the percentage of cell viability by deducting the blank control absorbance value from the measured value, dividing the result by the absorbance of the THF solvent control value and multiplying by 100%Calculate the half maximal inhibitory concentration values using graphing software.
The reported IC 50 is the average of all IC 50 values collected on at least three different days, and the error value is the standard deviation. The data received from the MTT assay is analyzed to evaluate the cytotoxicity of the compound. The measurements should be performed on a range of concentrations that will produce a graph displaying an even point distribution at least three points on each plateau and three points to define the slope.
Insufficient measurements at high concentrations or low concentrations will impair the nonlinear regression fit of the curve required for determination of IC 50 values, and as a result diminish their accuracy. Relative IC 50 values as determined by the nonlinear regression fit correspond to the concentrations leading to 50%of the cell growth inhibition possible by the compound tested. This is calculated as the middle point between the maximal and minimal cell viability measured, which are not necessarily 100%and 0%respectively.
When inspecting the results of the experiments described in the protocol. It is clear that cisplatin as well as the titanium and vanadium complexes are highly active. It is also noticeable that the closer the maximal inhibition value of a given compound is to 100%the closer the relative IC 50 value is to the absolute one when comparing the IC 50 values among the different compounds.
The titanium four complex has the highest cytotoxicity and its IC 50 values are lower than those of cisplatin and the Vanadium five complex. However, the free ligand exhibits markedly reduced activity since following addition of the ligand at different concentrations. The cell viability does not drop as significantly as it does for its complexes.
This assures that the high anti-cancer activity of the complexes relies on the metal centers While attempting this procedure. It's important to keep sterile conditions at all times. After watching this video, you should have a good understanding of how to evaluate the cytotoxicity of your anti-cancer compounds by the M-T-T-S-A.
