Anti cd54 fitc

Cell surface expression of leukocyte adhesion molecules was measured using flow cytometry. In brief, cells were seeded in 1 μg/cm² collagen I (Gibco) coated T25 flasks and were grown to confluence in M199 growth media. Once cells reached 100% confluency, media was replaced with fresh M199 growth media only, or M199 growth media containing either 0.5 ng/mL IL-1β or 0.5 ng/mL TNFα. Cells were treated for 37°C for 24 h, and were detached using EDTA-based Versene (Gibco). The cell suspension was adjusted to final cell concentration of 1x10⁶ cells per mL in cold FACS buffer (1% FBS in PBS). Fluorochrome-conjugated antibodies anti-CD54-FITC (cat#353108, Biolegend) and anti-CD106-PE (cat# 305806, Biolegend) were added to cells at previously optimised titrations [10 (link)]. Cells were incubated for 10 min on ice, and were washed twice in 1 mL of cold FACS buffer followed by centrifugation at 400x g for 10 min. The cell pellet was re-suspended in 100μl of FACS buffer. The stained cells were acquired using an Accuri C6 flow cytometer (BD Bioscience). Gating strategy was done as previously described in [10 (link)], where 7AAD was used to stained dead cells and define the live-cell gate.

RPMI 1640, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from GE healthcare (PAA, Pasching, Austria); Anti-CD54-FITC was purchased from Biolegend (San Diego, CA, USA). Anti-CD13-FITC, CD33-PE, anti-CD15-PE, and anti-CD11b- PE were purchased from Affymetrix (eBioscience, San Diego, CA, USA). Primary antibodies for p-MEK1/2, total MEK1/2, p-P44/42 MAPK, total P44/42 MAPK, p-JNK, total JNK, p-P38, total P38, ICAM-1, p-P65, and total P65 were all obtained from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies for p-STAT1 and total STAT1 were purchased from Beckton Dickinson (BD Biosciences, San Jose, CA, USA). Anti-β-actin and horseradish peroxidase coupled secondary antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Horseradish peroxidase coupled secondary antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).
Dimethyl sulfoxide (DMSO), all-trans retinoic acid (ATRA), and celastrol were all purchased from Sigma-Aldrich (St Louis, MO, USA). ATRA and celastrol were dissolved to 100 mM in DMSO, stored at −20°C, and used within three month. ATRA and celastrol was further diluted with culture medium (for cell culture experiments) or PBS (for animal experiments) just before use.

The antibodies used to identify the phenotype of CAR-T cells including anti-Human CD3 (APC-Cy7), anti-Human CD4 (PE), anti-Human CD45RO (PE-Cy7), anti-Human CD62L (FITC), and anti-Human CD8 (FITC) antibodies, as well as corresponding mouse IgG controls and anti-CD54 (FITC), were all purchased from BioLegend. ICAM1 expression levels on the surface of human tumor cells and ICAM1-CAR transduction efficiency were evaluated via purified ICAM1-scFv-Fc and ICAM1 ECD-Fc, respectively. The transduction efficiency of control CAR-T (anti-CD19) was evaluated via Myc-tag mouse mAbs staining (Cell Signaling Technologies). Anti-mouse IgG-FITC (BioLegend) was used to label the Fc of ICAM1 ECD-Fc. Fluorescence was assessed using a BD Fortessa flow cytometer and analyzed using FlowJo 10.6.0 software.