Non-interactive video download link (mp4).

The script that has been used to create the video shown above can be downloaded as excel script file.

1 Each team will receive two T25 cellculture flasks: one flask with TNCB1 and one with TNBC2. Take the flasks from the CO2 incubator and inspect the cells under the light microscope:
  • 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?
Write this down in your lab journal! 
2 Sterilize the suction needle and remove the medium from the flask carefully without touching the cells.
3 Pipet 2 mL of sterile PBS into the flask and slowly rinse the cells by swirling.
4 Sterilize the suction needle and remove the PBS from the cell culture flask.
5 Pipet 1 mL of 0.25% Trypsin and swirl solution over the culture surface. Put your cells back in the incubator for 1-2 minutes (TNBC2) or at least 5 minutes (TNBC1)
6 After  incubation, inspect the cells to see if cells have detached (tapping may help to detach cells further), otherwise place the flask in the incubator for another 1-3 minutes.
  • 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.
10 Based on your cell count per mL, calculate the volume of cells you need to dilute in order to seed out 1500 cells/well (TNBC1) and 1000 cells/well (TNBC2) in a final volume of 100 µL/well. NOTE: Prepare a cell suspension enough for 40 wells per plate for each celline although you only need 24 per plate for each celline! This is to make sure that you always have enough cell suspension.
  • 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!
IMPORTANT: One T25 flask of TNBC-cells is used to plate the 96-wells for IF and SRB! So each team receives one T25-flask with TNBC1 and 1 T25-flask with TNBC2, which are used for both 96-wells plates.
12 Incubate the cells overnight at 37°C and 5% CO2.
  • 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.

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  • Skill levelIntroduction video
  • CategoryCell biology

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This video is created by Leiden Academic Centre for Drug Research (LACDR), Faculty of Science at Leiden University under a open Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. When using this video in its original version please refer to When adapting the video, mention the source ‘adapted based on the original version that is created by the team’. It is not allowed to use the video for commercial purposes without consultation with the creators. You can contact us via


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