Cell plating and exposure

• Are there (many) floating cells ?
• What is the percentage confluency in the flask?
• Describe the shape of the cells that are still attached to the bottom of the flask. Also write down if you see any irregular shaped cells and if so, how many (few or considerable amount).
• What is the color of the culture medium?

• Add 4 ml complete RPMI culture medium and resuspend cells by pipetting the cell suspension up and down several times to dislodge all clumps.
• Transfer the 5 mL cell suspension into a 15 mL tube. (Why would you do this?)

• Turn on the cell counter.
• Pipet 10 µl of the cellsuspension onto one side of a TC10 slide (the other side can be used for the 2nd celline).

• Label the counter slide: group, team, cell-line (not on the bottom!)
• Put the slide into the cell counter and Press Enter.
• Write down the cell counts in your labjournal.

• Prepare the dilution according to your calculation. Make sure to resuspend your cells before taking your calculated volume of cell suspension and diluting it with medium!
• Transfer 100 µL to each well in the plate indicated in the colored area. Resuspend your cells before pipitting. 
• Add PBS (without cells) to the wells that surround the wells in which you plated your cells!

• Hand back remaining solutions to the assistent (Medium, PBS ect.).
• Discard the rest of the cells and other empty tubes and T25 flasks into blue bins.

• Empty waste bin into the blue container.
• If necessary, replace empty pipette tip boxes.
• If necessary, add 5 ml pipets to holder.
• Clean needle by flushing with 70% EtOH followed by mq.
• Clean the workspace with 70% EtOH.
• Shut off gas and vacuum system.
• Shut the window and turn off the flow cabinet.

After seeding the cells, the 96-wells plates will be placed in the incubator, why?

1. The cells can rest and recover.
   Correct = yes
2. You can prepare the next experiment.
   Correct = no
3. The cells can attach to the bottom of the plate.
   Correct = yes
4. The time planned for this part of the experiment is limited during the practical. Therefore, it is better to continue the next day.
   Correct = no

Feedback if correct: The seeding process is very stressful for cells. So, it is better to let the cells rest before continuing with the experiment. Also, the cells need some time to attach to the bottom of the plate, the cells are floating in the medium after seeding.

Feedback if b: This is a pleasant incidental, but not the main reason why the cells are placed in the incubator

Feedback if d: This is partly true but not the main reason why the cells should be placed in the incubator.

Before seeding your cells, it is necessary to resuspend your cell suspension. Why is this resuspension required?

1. If you don’t resuspend the cells will clump together, clogging the pipet this and therefore resulting in inaccurate volumes in the wells.
   Incorrect: The cells will indeed clump together, but this won’t clog the pipet tip.
2. If you skip this step, the cells will not be homogenously distributed resulting in some wells containing more cells than others.
   Correct! In experiments, all your samples need to have the same conditions except your variable (In this experiment: the inhibitors). For experiments where you want to research the effect of certain conditions on the proliferation of your cells, it is extremely important to begin with the same number of cells. When your suspension is homogenously distributed, you will prevent false positives and negatives.
3. The cells will all be attached to the tube and not in suspension.
   Incorrect: Will all cells attach to the tube?
4. All of the above.
   Incorrect. There is a grain of truth in each answer, but one of the answers is the main reason of the resuspension step.

Enter the cell density and calculate how much cell suspension you need to prepare sufficient diluted solution for 1500 TNBC1 cells in 100 µL per well (use TNBC1 as example).

• Total # wells needed: _a_
• Total diluted solution needed:_b_ mL
• Measured cell density: _c_×10⁶ cells/mL

_d_ µL cell suspension required to obtain the diluted solution.
Incorrect answer
Feedback id a is not 40: Look at the plate-layout in the next step. How many wells do you need to plate your cells?

Feedback if b is not 4: How much volume should each well contain? So what is the total for all 40 wells?

Feedback if d is not 15000*4/(c*10^6): You did not correctly calculate how much cell suspension you need to take for a dilution with a total volume of 4 mL.

Feedback if correct: Given your concentration of [mathresult|c] ×10⁶ cells/mL you should prepare a dilution by taking [mathresult|d] µL and filling up with medium to a total of 4 mL.

Why is it necessary to add PBS to your flask after removal of the culture medium?

1. To remove all dead cells.
   Correct = no
2. To remove any waste products excreted by the cells.
   Correct = no
3. To prevent the inactivation of trypsin by proteins present in the cell culture medium.
   Correct = no
4. To detach cells before adding trypsin.
   Correct = no

Feedback if correct. PBS is needed to wash the cells and get rid of medium components like serum, which inactivates trypsin. Without a PBS washing step will not/hardly detach from your flask.

Feedback if a: PBS is not needed to remove dead cells, you can simply discard to medium to do so.

Feedback if incorrect: No try again. One option is correct.