3.1 Cell resurrection
Fibroblast and macrophage cells will be taken from Faculty of Medicine, UniSZA. The fibroblast cell are used for the purpose of representing human normal cells while the macrophage cells are for representing human immune cells in the effort to detect the cell response in the body when this compound is introduced. The cells will be resurrected from cyropreservation. The cryovials containing frozen cells will be put into a 37°C water bath for thawing process. The vials will be gently swirled in the 37°C waterbath for less than 1 minute until there is just a small bit of ice in the vial.
Cryovials will be transferred from water bath into a laminar flow hood. The outside of the hood is made sure to be sterilized with 70% ethanol. Thawed cells will be transferred dropwise into a centrifuge tube containing prewarmed media and fetal calf serum.
The cell suspension will then be centrifuged at 200 rpm for 5 minutes. After centrifugation is completed, cells will be transferred into flask gently with DMEM growth medium. The cells will be incubated in 5% CO_2 incubator.
3.2 Cell culture
Preparation of growth medium will be done prior to culturing. 500 ml of pre-made Dulbecco’s Modified Eagle’s media (DMEM) will be supplemented with 50 ml (10%) of fetal calf serum, 100 U/ml penicillin/streptomycin. This media is for culturing fibroblast cells. For the macrophage cells, Roswell Park Memorial Institute medium (RPMI1640) will be used as growth medium. The media powder will be dissolved in 1 L of distilled water in a beaker. While it is stirred on a hot plate, sodium bicarbonate will be added. Then, the pH of the media will be adjusted to 7.4. The RPMI1640 will also be supplemented with 100 ml (10%) of fetal calf serum, and 200 U/ml penicillin/streptomycin.
Resurrected cells will be passaged into new culture flasks. Two T175 flasks will be filled with growth culture media. Next, cells are taken out from incubator and the used culture media will be disposed. The cells will then be trypsinized with 0.25% trypsin-EDTA solution to detach the cells from the surface of the culture flask. Growth media will be added to stop trypsin reaction with the cells. Then, the total amount of medium in the flask will be splitted into the two new culture flasks and followed with incubation.
Cell maintenance will be done by replacing fresh medium twice every week. Cultures with confluency more than 95 % will be ppassage into new culture flasks while cultures with 75-95 % confluency will be used for the experiment. Cell confluency is to be tested with trypan blue exclusion test. Cells will be seeded in 96 well plate one day prior to treatment of thiosemicarbazide.
3.3 Compound preparation
The thiosemicarbazide compound will be prepared for a stock solution with the concentration of 10 mM by the addition of complete media and DMSO. A serial dilution with varying concentration of 0.1 mM, 0.01 mM, 0.001 mM, 1 X ?10?^4 mM, 1 X ?10?^5 mM, & 1 X ?10?^6 mM will be prepared from the stock.
3.4 Cell viability assay
The cell viability will be assesed by MTT assay where the protocol starts from trypsinization of the cells. This is to achieve the first objective of the study. The cells will be splitted and seeded into 96 well plate. Then, the cell is left until the confluency is 90-100 %, and it will be ready for use. TSC-Ni treatment will be added to the wells according to the desired concentrations with 5 replicates each. Cells will be incubated for 24 hours in 5% CO2 incubator. This is to allow reaction between the compound and the cells to take place.
MTT solutions will be prepared by dissolving 1 mg MTT in 1 mL PBS solution and the molarity will be at 2.41 mM. 20 µL of the solution into each well together with 160 µl of growth media and followed by incubation for 4 hours. After discarding media, 100 µl of DMSO will be added to the well. Then, the well will be covered with aluminium foil and mildly shaked for 15 minutes. The reading of the plate will be done using fluorescent reader in 570 nm absorbance.
To address the second objective, a list of targeted proteins will be screened using Swiss target prediction (www.swisstargetprediction.ch) and a simulation of docking of the proteins with TSC-Ni will be done on Systems dock (systemsdock.unit.oist.jp).
3.5 Assessment on mode of cell death
AO/PI staining protocol will be used to assess mode of cell death which is the third objective. Firstly, cells should be harvested into centrifuge tube. Cells will be removed from incubator and trypsinization will take place to detach the cells. To let the cells react with trypsin, cells will be reincubated for 10 mins. Then, trypsinization will be stopped by adding growth media into the flask. The cells will be centrifuged at 1500 rpm for 5 minutes.
Next, the cells are to be suspended in PBS. In a separate centrifuge tube, aliquot some cell suspension and add AO/PI with ratio 5:1.
AO will stain both viable and unviable cells thus PI stain is used to counterstain the unviable cells, enabling to differentiate between living cells and dead cells. Then, properties of membrane permeability and mode of cell death will be determined.
3.6 Statistical analysis
Analysis of cytotoxic activity will be assessed by using two-tail T test. Number of cell viable cells stained by MTT solution in treated cells will be compared with control (untreated cells), and the reading of absorbance on microplate reader will be taken as the measure of cell viability. results will be expressed as mean +/- standard deviation with each sample being tested in at least triplicate. Plotting a curve with percentage of cell viability (on y-axis) against varying concentration of TSC-Ni (on x-axis) will determine the IC_50 value of the compound. Two-tail T test analysis will be done at 95% significant level.