PROTOCOL

Detailed T cell TEM Chemotaxis Protocol

Materials

  • Freshly isolated blood
  • Lympholyte®-H Cell Separation Media (Cedarlane CL5010)
  • Dynabeads® Human T-activator CD3/CD28 (Life Technologies 11131D)
  • RPMI 1640 Medium (Life Technologies 11875-085)
  • Dialyzed FBS (Omega Scientific FB-03)
  • HUVEC (LONZA C2517A)
  • EGM-2 MV Bullet Kit (LONZA CC-3202)
  • Collagen Type I Rat Tail (BD Biosciences 354236)
  • Fibronectin (Sigma Aldrich F1141)
  • Acetic Acid (0.02N) – used in Collagen coating step
  • D-PBS -/- (w/o Ca2+, Mg2+, Life Technologies 10010) – used in Fibronectin coating step
  • D-PBS +/+ (with Ca2+, Mg2+, Life Technologies 14040) – used in monolayer wash step
  • SDF-1a (RnD systems 350-NS-050)
  • IncuCyte™ ClearView 96-Well Cell Migration Plate (Essen 4582 or 4599)

Create HUVEC Monolayer

  1. Prepare extracellular matrix coating of either 50 µg/mL Collagen diluted with 0.02N Acetic Acid or 5 µg/mL Fibronectin diluted with D-PBS (-/-).
  2. Pipette 150 µL of coating solution into the reservoir. Gently place the insert into the reservoir and pipette 20 µL of the fibronectin, or collagen, solution into the insert.
  3. Incubate for 1 hour at ambient temperature.
  4. During incubation, harvest and count HUVEC cells and prepare a cell seeding stock of 100,000 cells/mL in full growth medium (EGM-2).
  5. Aspirate the coating from the reservoir plate and replace with 200 µL of D-PBS (-/-) and gently return the insert into to the reservoir plate.
  6. To the insert, directly add 60 µL D-PBS (-/-) to the inserts containing coating, then aspirate the entire volume.
  7. Immediately seed HUVECs using a multi-channel pipette into every well of the insert plate (60 µL per well, 6,000 cells per well)

    Calculation: 100,000 cells/mL x 0.06 mL = 6,000 cells per insert well.

  8. Allow the HUVECs to settle at ambient temperature on a level surface for 15 minutes.
  9. Place the Chemotaxis plate containing HUVEC cells at 37°C and incubate for 24 hours.

Initiating TEM

  1. After HUVEC monolayer has formed, gently wash the monolayer 2x with D-PBS (+/+), using partial washes.

    Note: It is important not to disrupt the HUVEC monolayer. It is recommended to gently remove about half of the growth medium then add 60 µL D-BPS for both washes. At the final wash step, remove as much of the medium/D-PBS as possible without disrupting the monolayer.

  2. Prepare T-cell cell seeding stock at 83,333 cells/mL in defined EBM-2 (EBM media + 2% FBS + hydrocortisone + ascorbic acid + heparin).
  3. Using a manual multi-channel pipette and reverse pipetting technique, seed cells (60 µL per well, 5,000 cells per well) into every well of the insert plate, being careful not to disrupt the HUVEC monolayer.

    Calculation: 83,333 cells/mL x 0.06 mL = 5,000 cells per insert well.

  4. Centrifuge the chemotaxis plate for 3 minutes at 50 x g in order to quickly bring the T-cells to the monolayer surface. Alternatively, if centrifugation is not possible, allow the T-cells to settle on the HUVEC monolayer at ambient temperature for 45-60 minutes.
  5. Using a manual multi-channel pipette, add 200 µL of the chemoattractant and control medium to the appropriate wells of the second reservoir plate.
  6. Carefully transfer the insert plate containing the cells into the pre-filled second reservoir plate containing medium ± chemoattractant.
  7. Place the IncuCyte™ ClearView cell migration plate into the IncuCyte ZOOM® instrument and allow the plate to warm to 37°C for at least 15 minutes. After 15 minutes, wipe away any condensation that remains on the outside of the plate lid or bottom of the reservoir.
  8. In the IncuCyte ZOOM® software, schedule 24 hour repeat scanning (10x) for every 30 minutes.

    NOTE: This schedule is only for a scanning a single plate. Fewer scans times will be required if scheduling multiple plates

    • Objective: Ensure 10x objective is installed
    • Vessel Type: Select “ClearView Cell Migration”
    • Channel Selection: Select “Phase”
    • Scan Mode: Select “Chemotaxis (Top/Bot)” scan type and desired Scan Pattern
    • Note the IncuCyte™ instrument estimates a scan time of 20 min per plate (phase only); however, the actual scan time can take longer.

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