Protocol 012 - Plating, Drug Dilutions, and Treatment Schema Using Chordoma Cell lines in a 96-well plate for Cytostatic Compounds

Wash cells in T75 flask with 8mL of 1X PBS, swirl PBS to wash cells, and aspirate.

Add 3mL of trypsin to the T75 flask. Swirl trypsin for ~5-10 seconds to fully coat cells and then quickly aspirate. Place in incubator for ~3-5 minutes or until cells have detached without hitting the vessel.

Pipet 8mL of appropriate media to the trypsinized cells in the T75 flask. Pipet up and down to facilitate dissociation to a single cell suspension. Transfer cell suspension to a 15mL conical and spin at 250xg for 2.5 minutes.

While the cells are spinning, pipet 100uL of 1X PBS into the outer wells of the 96-well plate (A1 -> A12, H1 -> H12, B1 -> G1, B12 -> G12). This PBS is used to act as a buffering layer to decrease the edge effect of sample wells

Aspirate supernatant and resuspend cells in 8mL of appropriate media. Immediately remove 20uL from cell suspension and transfer to a 1.5mL microcentrifuge tube. pipet in 80uL of 0.4% trypan blue, mix thoroughly, and then remove 10uL to pipet into hemocytometer.

Count cells in the 4 outermost squares and input number into benchling template with correct cell line chosen.

96-well plate seeding densities per cell line for cytostatic compounds:

Make cell dilutions for seeding densities above. For 1 drug, 30-wells will be used (30wells * 100uL cell suspension = 3mL)

Pour cell suspension into a 25mL reservoir and pipet 100uL of cell suspension to each well

Pipet 1X PBS into the outer wells of the plate (A1 -> A12, H1 -> H12, B1 -> G1, B12 -> G12) if this was not done in step #4 f this protocol

Plate in incubator at 37°C and 5% CO2

Treat ~36 hours later

Assay duration per cell line.

As presented below in the "96-well Plating and Treatment Schema", a single cell line is plated in 30 wells from B2:D11. A second cell line is plated in 30 wells from E2:G11. Cell line names are labelled to the left boundary of the plate in the center of the 3 rows where said cell line is plated, presented as "Cell Line A" and "Cell Line B". The grey borders represent wells that are filled with sterile 1X PBS, ddH₂O, or media to remedy the edge effect.

In general, there will be 2 cell lines/plate and 1 drug/plate. Drug name should be labelled on the right side of the plate centered with the rows in which the drug is added.

The drug will then be serially diluted from B11:G3 by 1/3. Drug concentration/well is outlined below the 96-well plate image. Serial dilutions are covered in Fig. 2. The standard starting drug concentration, from B11:G11, will be 10uM. This may deviate from 10uM to 5uM, 3uM, or 1uM, depending on the drug being used. The date treated should be written on the bottom edge of the 96-well plate along with a checkbox signifying the date was verified and the plate is in a constant state until reading, no other changes will occur.

Plates are numbered in the bottom right hand corner for ease of recording and identifying. When using public equipment it is important to use identifiers only known to the lab itself. Experiments should be named "Project Name_Experiment # -- Plate # -- Date". It is good general practice to take a picture of the plates on the date of reading to supplement the ELN Entry.

For every 30 well dose curve with 100uL of drug and media in the wells 6mL of CTG is needed. Warm needed + 1mL for excess. Remove CTG reagent from the -20° and let warm to room temperature in the dark. A drawer is sufficient. DO NOT ADD ADDITIONAL HEAT OR LEAVE IN THE LIGHT.

Remove 96-well white walled plate from incubator and let cool to room temperature. Normally this takes around 15 minutes.

Thoroughly mix CellTiter-Glo 2.0(CTG) and dispense in 25mL reservoir.

Using a 10 channels of a 12-channel multichannel pipet, pipet 100uL of CTG reagent to each row, carefully, to avoid bubble.

After addition to all necessary wells, place on plate shaker at 600rpm for 2 minutes, covered to protect from light.

Remove plate and store at RT in the dark for 1 hour.

Remove plate and read on the Biotek Cytation 5 plate reader using the Chordoma Foundation CTG protocol.