Figure 2
Figure 2. Adhesive interactions of WT and αL-I306A (KI) cells with ICAM-1 in vitro. (A) Cell adhesion of splenocytes to ICAM-1 and VCAM-1 substrates studied using a V-bottom-well plate. Cell suspensions were added to the plates and immediately centrifuged at 200g for 15 minutes to remove unbound cells. (B) Binding of soluble ICAM-1–Fc to WT and KI splenocytes. Bound ICAM-1–Fc was detected with indirect immunofluorescent cytometry using fluorescein isothiocyanate (FITC) anti–human immunoglobulin G (Fc-specific) antibody. (C) Transmigration of WT and KI splenocytes toward a CCL21 gradient through mock-, ICAM-1–coated, and VCAM-1–coated permeable inserts was examined using a modified Boyden chamber assay with a Transwell system (Corning Inc). (A-C) Data are expressed as the mean ± SEM of triplicates from 4 independent experiments. (D-I) 2D migration of T lymphocytes on ICAM-1. (D) 2D tracking of WT and αL-I306A (KI) TCM migrating ICAM-1/CXCL12 substrates. Each track (red) represents migratory paths of individual WT (n = 20) and KI (n = 20) over a 25-minute period. Mean displacement (E), mean velocity (F), and meandering index (G) of laterally migrating TCMs on ICAM-1/CXCL12 substrates were obtained from analysis of live-cell imaging. (E-G) Data are expressed as mean values ± SEM. (H) Representative confocal images of T lymphocytes migrating on ICAM-1/CXCL12 stained for actin (Alexa 488) and αL integrin (cyanin 3) are shown. White bars represent 10 μm. Confocal imaging was performed with a Radiance 2000 Laser-scanning confocal system (Bio-Rad Laboratories) using an Olympus BX50BWI microscope outfitted with a 100×/1.0 numeric aperture water-immersion objective coupled to a photomultiplier tube (PMT) detection system. Imaging medium was PBS. Images were acquired with BioRad2000 Version 2 software and subsequently processed with OpenLab Version 3.0 software. (I) The polarization index16 of T cells from randomly selected fields. Thick horizontal bars indicate mean values. (A-C,E-F,I) Data are expressed as the mean values ± SEM. Two-tailed Student t test was used for statistical analyses. Statistical significance was defined as *P < .05, **P < .01, or ***P < .001.

Adhesive interactions of WT and αL-I306A (KI) cells with ICAM-1 in vitro. (A) Cell adhesion of splenocytes to ICAM-1 and VCAM-1 substrates studied using a V-bottom-well plate. Cell suspensions were added to the plates and immediately centrifuged at 200g for 15 minutes to remove unbound cells. (B) Binding of soluble ICAM-1–Fc to WT and KI splenocytes. Bound ICAM-1–Fc was detected with indirect immunofluorescent cytometry using fluorescein isothiocyanate (FITC) anti–human immunoglobulin G (Fc-specific) antibody. (C) Transmigration of WT and KI splenocytes toward a CCL21 gradient through mock-, ICAM-1–coated, and VCAM-1–coated permeable inserts was examined using a modified Boyden chamber assay with a Transwell system (Corning Inc). (A-C) Data are expressed as the mean ± SEM of triplicates from 4 independent experiments. (D-I) 2D migration of T lymphocytes on ICAM-1. (D) 2D tracking of WT and αL-I306A (KI) TCM migrating ICAM-1/CXCL12 substrates. Each track (red) represents migratory paths of individual WT (n = 20) and KI (n = 20) over a 25-minute period. Mean displacement (E), mean velocity (F), and meandering index (G) of laterally migrating TCMs on ICAM-1/CXCL12 substrates were obtained from analysis of live-cell imaging. (E-G) Data are expressed as mean values ± SEM. (H) Representative confocal images of T lymphocytes migrating on ICAM-1/CXCL12 stained for actin (Alexa 488) and αL integrin (cyanin 3) are shown. White bars represent 10 μm. Confocal imaging was performed with a Radiance 2000 Laser-scanning confocal system (Bio-Rad Laboratories) using an Olympus BX50BWI microscope outfitted with a 100×/1.0 numeric aperture water-immersion objective coupled to a photomultiplier tube (PMT) detection system. Imaging medium was PBS. Images were acquired with BioRad2000 Version 2 software and subsequently processed with OpenLab Version 3.0 software. (I) The polarization index16  of T cells from randomly selected fields. Thick horizontal bars indicate mean values. (A-C,E-F,I) Data are expressed as the mean values ± SEM. Two-tailed Student t test was used for statistical analyses. Statistical significance was defined as *P < .05, **P < .01, or ***P < .001.

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