Approximately 3.0 × 104 CTLs cells transfected with the indicated plasmids were resuspended in imaging medium RPMI–HEPES without phenol red (Thermofisher Scientific, Waltham MA) and deposited onto a 8-well chambered coverglass (Lab-Tek II, Nunc) coated with 5ug/ml anti-CD3 (clone OKT3, BD) and anti-CD28 (BD) antibodies and 10ug/ml recombinant human ICAM-1/CD54-Fc-chimera (R&D Systems, Minneapolis MN). Cells are imaged at 10–20 frames/sec rate using a Leica DMi8 microscope equipped with ring-TIRF iLas-2 system, a dualCam with two Hamamatsu EM-CCD cameras and an environmental control system set to 5 % CO2 and 37°C. Fluorophores were excited with solid state 488 nm and 561nm lasers, TIRF angle was set to 90 nm depth. Images were analyzed and processed using FIJI software 41 (link). Quantification of the Rab11a vesicles dwell-time was performed using the TracMate function of FIJI software.
Recombinant human icam 1 cd54 fc chimera
Manufactured by R&D Systems
Recombinant human ICAM-1/CD54-Fc-chimera is a protein consisting of the extracellular domain of human ICAM-1 (Intercellular Adhesion Molecule-1) fused to the Fc region of human IgG1. It is produced in a mammalian cell expression system.
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Lab products found in correlation
Phenol red, by Thermo Fisher Scientific (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Em ccd camera, by Hamamatsu Photonics (1 mentions)
Lab tek 2, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by BD (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Sheep anti mouse igg hrp, by Cytiva (1 mentions)
Goat anti rabbit ig hrp, by Agilent Technologies (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
P src, by Cell Signaling Technology (1 mentions)
Anti cd11a, by Abcam (1 mentions)
Fibronectin, by R&D Systems (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Recombinant vcam 1, by R&D Systems (1 mentions)
Pzap 70, by Cell Signaling Technology (1 mentions)
α β tubulin, by Cell Signaling Technology (1 mentions)
3 protocols using recombinant human icam 1 cd54 fc chimera
1
Imaging and Analysis of Rab11a Vesicles
2
Immunoblotting of T-cell signaling
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Primary antibodies: Mouse monoclonal antibody (Ab) and affinity-purified goat polyclonal Ab, both raised against human PTPN22, were purchased from R&D Systems. Antibodies to Vav1 (C-14), ZAP-70 (1E7.2), Lck (3A5), Csk (C20), were from Santa Cruz Biotechnology. Rabbit polyclonal antibodies: p-Src (Tyr416), p-ZAP70 (Tyr319/SykTyr352), p-ZAP70 (Tyr493/Syk-Tyr526), p-Erk (Thr202/Tyr204) (197G2), α/β-tubulin, β-actin (Cell Signalling Technology), p-Vav1(Tyr174) (EP5107) (Abcam), and anti-PAG (PAG-C1) and anti-CD11a (LFA-1). mAb38 was a kind gift of Dr Nancy Hogg. Secondary antibodies for immunoblotting detection: Sheep anti-mouse IgG-HRP (Amersham), goat anti-rabbit Ig-HRP (DAKO), mouse monoclonal light chain specific anti-goat IgG (Jackson Laboratory), goat anti-mouse IgG, goat anti-rabbit IgG, donkey anti-mouse IgG, donkey anti-rabbit IgG, donkey anti-goat IgG, and IgG1, IgG2b
Zenon® antibody labelling kits were all from Life Technologies. Integrin ligands: Recombinant human ICAM-1/CD54 Fc chimera, recombinant murine ICAM-1/CD54 Fc chimera, recombinant VCAM-1 and fibronectin (R&D Systems).
Zenon® antibody labelling kits were all from Life Technologies. Integrin ligands: Recombinant human ICAM-1/CD54 Fc chimera, recombinant murine ICAM-1/CD54 Fc chimera, recombinant VCAM-1 and fibronectin (R&D Systems).
3
3D Migration Assay of VAL Cells
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Cell line and cultures VAL cells were cultured in medium supplemented with 10% heatinactivated Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin, 1% GlutaMAX. 1% NEAA, 1% Sodium-Pyruvate, and 50 µM β-mercaptoethanol. CFP+ cells were cloned as described previously 18 .
3D migration Migration assays of VAL cells were performed using the 3D chamber µ-Slide from Ibidi as described in Antonello et al. (submitted). Briefly, cells were embedded in a collagen matrix formed by 1.6 mg/mL PureCol (Collagen, Sigma-Aldrich), 0.36% PBS supplemented with 0.36% FBS, 0.036% P/S, 1.5 μg/mL recombinant human ICAM-1/CD54 Fc chimera (R&D systems) at 4° C. The temperature was slowly raised over 45 min to 37°C to induce a homogeneous collagen polymerization. Complete medium was added to both side reservoirs. After 24 hours 15 µL of 400 nM CXCL12 were added to one of the reservoirs and time-lapse video microscopy was performed for 6 hours at 20 seconds time intervals using an ImageXpress® (Molecular Devices) high throughput microscope.
3D migration Migration assays of VAL cells were performed using the 3D chamber µ-Slide from Ibidi as described in Antonello et al. (submitted). Briefly, cells were embedded in a collagen matrix formed by 1.6 mg/mL PureCol (Collagen, Sigma-Aldrich), 0.36% PBS supplemented with 0.36% FBS, 0.036% P/S, 1.5 μg/mL recombinant human ICAM-1/CD54 Fc chimera (R&D systems) at 4° C. The temperature was slowly raised over 45 min to 37°C to induce a homogeneous collagen polymerization. Complete medium was added to both side reservoirs. After 24 hours 15 µL of 400 nM CXCL12 were added to one of the reservoirs and time-lapse video microscopy was performed for 6 hours at 20 seconds time intervals using an ImageXpress® (Molecular Devices) high throughput microscope.
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