Tnf 1α

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.