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1. Generation of reproducibly sized tumor spheroids
- Collect the Diffuse Midline Glioma (DMG) H3K27M-mutant neurospheres (NS) and centrifuge at 170 x g for 10 minutes (min) at room temperature (RT).
- To break up the NS, incubate 500 µL of the accutase solution for 3 min at 37 °C.
- Neutralize the accutase solution with tumor stem cell (TSM) medium7 and centrifuge the cell suspension at 355 x g for 5 min at RT.
- Resuspend the cell pellet in 1 mL of TSM medium and then cont the cells using a cell counting chamber.
- Dilute the cell suspension to obtain 2.5-5 x 103 cells/mL and seed 100 µL/well into ultra-low attachment (ULA) 96-well round-bottom plates (see Table of Materials). Use a proper cell density to obtain individual NS of ~300 µm diameter, 4 days after cell seeding (250-500 cells/well for highly aggressive glioma cells).
- Visually confirm the NS formation by using an inverted microscope 4 days after cell seeding.
2. Preparation of the antibody labeling reagent (ALR)/antibody complex and setup for the migration assay
- Consider the number of wells to analyze and calculate the volume needed for each reagent. Include also the wells needed for the negative controls. Check the NS visually using an inverted microscope before starting.
- Use flat-bottom 96-well plates. Perform the coating. For this study, the basement membrane matrix (BMM) is used as a thin coating.
- Once the coating is ready, remove the excess BMM coating with a p200 tip, placing the tip on the edge of the well and avoiding touching the bottom. If working with multiple wells, use a multichannel pipette.
- Cut a p200 tip, take 50 µL of the cell medium + NS from each selected well, and transfer it to a coated flat bottom well. Check the presence and the position of the NS in each well visually.
NOTE: Each NS must be centrally located in the well. Avoid leaning on the tip on the edge of the well during the transfer but drop the medium centrally in the well without touching the bottom. For highly migratory cells, consider a higher number of replicates than the standard three replicates. This is because when NS sits too close to the edge of the well, the migrating cells may cover a smaller area of the well. - Add 100 µL of sterile water to the ALR to rehydrate the reagent (final concentration = 0.5 mg/mL). Pipette to mix the solution. NOTE: The reagent is light-sensitive; therefore, keep in the dark. Aliquot the leftover reagent and store at -80 °C (avoid freezing and thawing).
- Mix the antibody with ALR in the appropriate complete cell growth media in a round bottom multi-well plate or in an amber tube and protect from light. Prepare enough quantity to dispense 50 µL/well at 3x final assay concentration. Incubate at RT for 15 min.
- Dilute the background suppressor reagent (BSR) in TSM medium (or appropriate cell growth media for cell line of choice) at 1.5 mM (3x) to obtain at the end of the assay a final concentration of 0.5 mM.
NOTE: Reagent is light sensitive. See above for good handling procedures. - Gently add 50 µL of the BSR to each well.
- Gently add 50 µL of the ALR/antibody to each well. Wait 2 or 3 min to let the reagents mix and check visually using an inverted microscope to ensure that most of the replicate NS are centrally located in the well.
- Avoid the formation of bubbles and remove any existing ones by using a needle. Gently transfer the plate in the live-cell analysis instrument placed within the incubator at 37 °C, 5% CO2, 95% humidity.
3. Live-cell analysis instrument setting for image acquisition
- Scan the plates using the live-cell analysis instrument (for specifications, see Table of Materials) with scanning intervals starting from time point zero (t0) of the invasion and migration assays set up, respectively, after step 2.14. and 3.10. up to 96 h.
NOTE: Ensure to be able to dispose of the live-cell analysis instrument immediately after the starting of the invasion and migration assay. Depending on the tumor type, cells can start to invade or migrate from the NS already within 1 h from the assay setup. - On the live-cell analysis instrument software, select the option Schedule to Acquire. Click on the + tab and select the option Scan on Schedule.
- On the software window Create or Restore Vessel, click on the option New.
- Select the specific application in the live-cell analysis instrument for the invasion and migration acquisition. Select Spheroid scan type, 4x objective, Phase+Brightfield and Green image channels for the invasion assay. Select Dilution Cloning scan type, 4x objective and Phase and Green for the migration assay.
- Select the plate type and define the wells to be scanned by highlighting them on the plate map.
- Set up the scanning frequency (for the experiments in this protocol scanning frequency was 15 min for invasion and 30 min for migration).
- Click on Add to Schedule and start the scan.