Initiating TEM
- 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.
- Prepare leukocyte cell seeding stock at 83,333 cells/mL in appropriate assays medium.
- 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.
- 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.
- Using a manual multi-channel pipette, add 200 µL of the chemoattractant and control medium to the appropriate wells of the second reservoir plate.
- Carefully transfer the insert plate containing the cells into the pre-filled second reservoir plate containing assay medium ± chemoattractant.
- 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.
- 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