PROTOCOL

Chemotatic Transendothelial Migration General Protocol

Materials

  • Leukocytes (e.g. activated primary T-cells or neutrophils)
  • Endothelial cells (e.g. HUVEC or EA.hy9226)
  • Endothelial growth medium
  • Assay medium
  • Collagen Type I Rat Tail (BD Biosciences 354236)
  • Fibronectin (Sigma Aldrich F1141)
  • Acetic Acid
  • 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
  • IncuCyte™ ClearView 96-Well Cell Migration Plate (Essen 4582 or 4599)

See the table below for an example of the leukocyte and endothelial cells that have been validated with the TEM assay along with recommended growth and assay media.

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Create Endothelial Cell 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 endothelial cells and prepare a cell seeding stock of 100,000 cells/mL in full growth medium.
  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 endothelial cells in growth medium 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 cells to settle at ambient temperature on a level surface for 15 minutes.
  9. Place the Chemotaxis plate containing cells at 37°C and incubate for 24 hours.

Initiating TEM

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

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

  2. Prepare leukocyte cell seeding stock at 83,333 cells/mL in appropriate assays medium.
  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 endothelial 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 leukocytes to the monolayer surface. Alternatively, if centrifugation is not possible, allow the leukocytes to settle on the endothelial 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 assay 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.
Leukocyte Endothelial Cells Endothelial Growth Medium Assay Medium Coating
(20 µL top/150 µL bottom)
Seeding Density endothelial: leukocyte (cells/well)
Jurkat HUVEC EGM-2 EBM-2 + 2% FBS with #supplements 5 µg/mL Fibronectin or 50 µg/mL Collagen 6,000 : 5,000
Primary T-cells HUVEC EGM-2 EBM-2 + 2% FBS with #supplements 5 µg/mL Fibronectin or 50 µg/mL Collagen
EA.hy926 DMEM + 10% dialyzed FBS RPMI + 0.5% dialyzed FBS 50 µg/mL Collagen
Neutrophils HUVEC EGM-2 EBM-2 + 2% FBS with #supplements 5 µg/mL Fibronectin or 50 µg/mL Collagen

# Supplement includes gentamycin, hydrocortisone, ascorbic acid

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