μ slide 6 0.4 flow chambers

Forty-eight hours prior to the assay bEnd.3 endothelial cells were plated in tissue culture treated μ-Slide VI 0.4 flow chambers (ibidi). Twenty-four hours later, the endothelial monolayer was treated with 40 ng/mL TNF-1α (Peprotech), which upregulates expression on the bEnd.3 endothelial cells of adhesion molecules (such as ICAM-1 and VCAM-1) needed to support leukocyte TEM. Then 30–45 min prior to the assay the endothelial cells were treated with 1 μg/mL CXCL12, which promotes the rolling and adhesion of leukocytes on the endothelial cells. For the transendothelial assay, using a syringe pump, control, or mDia1 KD B-ALL cells (at 2 × 10⁶ cells/mL) were flowed onto the treated endothelial monolayer at 0.25 dyne/cm² for 5 min (accumulation phase), and then the flow rate was increased to 2 dyne/cm² (approximate physiological shear flow). Phase contrast and fluorescent images were acquired every 15–25 s using a 20X Phase-2 objective for 30 min long time-lapses using a Spinning Disk confocal microscope with environmental control (Intelligent Imaging Innovations) and Slidebook imaging software (Intelligent Imaging Innovations). Using similar criteria as previously described (11 (link), 29 (link)), a cell was scored as having undergone transendothelial when it lost its white phase ring in a step-wise process during the course of the time-lapse.

μ-Slide VI 0.4 flow chambers were obtained from Ibidi. Glass slides were cleaned by sonicating in 2% Hellmanex solution (Hellma, Mullheim, Germany) for 30 min and then rinsed with water. Subsequently, they were further treated with NOCHROMIX (Godax Laboratories, Inc) and concentrated H₂SO₄ for at least 6 hours, and then extensively rinsed with water. The glass slide was plasma cleaned and attached to the Ibidi chamber with double-sided tape around, forming a closed chamber with inlet and outlet. The buffer (20 mM HEPES / 150 mM NaCl, pH = 7.4) was injected to test whether leakage occurs. Small unilamellar vesicles (SUV) were then injected and incubated for 30 min to form the supported lipid bilayer. Any excess SUVs were washed out with buffer. Finally, YFP-PHGrp1 and PTEN were injected.