Invasion assay protocol

This is a detailed protocol we use for an inverted invasion assay, as used in Miller et al.

Before Starting

  1. Spray down and clean hood.
  2. Bring into hood: 20 uL tips, P20 pipette (bench 4), 1.5 mL tubes.
  3. Make 1.5 mL of each growth factor and inhibitor solution 3X concentrated in full serum media (10 tubes).
  4. Set centrifuge to 3C, open and close the lid. Make sure 96-well plate rotors are placed. Allow centrifuge to cool for 20 min.
  5. Remove everything from hood, place tubes in water bath.
  6. Warm PBS, full serum media and trypsin.

During

  1. Bring into hood: 200 μL tips (yellow), 12-channel pipette (bench 6), cell culture P200, 1000 μL tips (blue), P1000 pipette (bench 4), 2-15 mL falcon tubes (one into ice), large square bucket with ice, 10x DMEM (on ice), 1N NaOH (on ice), multichannel reservoirs, warm PBS, warm trypsin. Get a ViCell tube.
  2. Trypsinize a 10 cm plate of cells by washing once with 2 mL PBS and adding 2 mL trypsin.
  3. Once trypsinized, resuspend cells in 8 mL media in the warm falcon tube. Take 1 mL and count on ViCell. Sediment cells at 1260 rpm for 3 min.
  4. Resuspend cells at a concentration of 600k/mL.
  5. Place a reservoir on ice.
  6. Add 600 μL of 10x DMEM, then 108 μL of 1N NaOH to the falcon tube on ice and mix by pipetting.
  7. Using a 5 mL pipette tip, add 3.2 mL of acid extracted collagen and mix with the same tip by pipetting. Vacate the pipette, then wait for residual collagen to settle and vacate again.
  8. Bring black Nunc plates (4°C TC fridge) into the hood, and place one on ice.
  9. Using a 10 mL pipette, add 2 mL of cell solution to the collagen mix then immediately remove the entire solution and place in reservoir. Mix thoroughly using the same pipette.
  10. Immediately dispense 50 μL into each well of the 96-well plate and centrifuge for 8 min at 300×g (g’s are indicated when the speed has a * next to the number).
  11. Place plate in the incubator for 30 min.
  12. Put back everything except for single-channel P200 and tips (yellow). Keep full serum media in water bath.

After

  1. Bring warm inhibitor solutions and media into hood.
  2. Dispense 50 uL of appropriate solution into each well.
  3. Discard remaining inhibitor solutions; put media back in water bath. Wait 1 hr.
  4. Bring warm growth factor solutions and media into hood.
  5. Dispense 50 uL of appropriate solution into each well.
  6. Put away everything.

Next Day

Fixation

  1. Warm 4% PFA in PBS (frozen in back right of -20C) in water bath.
  2. Wait until 24 hr after growth factor stimulation.
  3. In fume hood, pour PFA into reservoir; bring 200 μL (yellow) tips and multichannel pipette.
  4. Get plate from incubator. Invert and blot plate on a pad of paper towels to remove media.
  5. Add 100 μL PFA to each well.
  6. Let sit at room temperature for 1 hr.
  7. Discard extra PFA, tips in chemical waste.
  8. In fume hood invert and blot plate on paper towels. Discard paper towels in chemical waste.

Staining

  1. Add 200 μL PBS to each well.
  2. Transfer 10 mL of 0.5% TX-100, 0.5% BSA in PBS (on 4°C non-TC shelf) to a 15 mL falcon tube.
  3. Add 2 μL DRAQ5 (in 4°C secondary box) and mix by inverting.
  4. Empty plate by blotting onto pad of paper towels.
  5. Place solution in reservoir and dispense 100 μL in each well.
  6. Incubate overnight at 4°C.
  7. Empty plate by blotting onto pad of paper towels.
  8. Add 100 uL 0.5% BSA in PBS (on 4C non-TC shelf).
  9. Repeat 7&8 once.
  10. Store at 4°C.