We routinely applied 2 × 105 PMNs per milliliter to cells. For antibody-mediated clustering of ICAM-1, sheep anti-mouse IgG-coupled dynabeads (M280; Invitrogen) were coated with mouse anti-human ICAM-1 antibody (BBIG-I1; R&D Systems) or immunoglobulin IgG1 (IgG1) control (MAB002; R&D Systems) overnight at 4°C according to the manufacturer’s protocol. To induce clustering, 1.5 × 106 antibody-coated beads/mL was added to a tumor necrosis factor α (TNFα)-stimulated HUVEC monolayer cultured in a 6-well dish and incubated for 15 minutes. For some experiments, antibody-coated beads (5 × 105/mL) were injected into the ibidi perfusion system containing HUVECs to induce ICAM-1 clustering on HUVECs under physiologic flow conditions. Alternatively, ICAM-1 was ligated with 15 μg/mL mouse monoclonal antibodies (R&D Systems, catalog no. BBIG-I1) for 30 minutes, followed by washing and ICAM-1 cross-linking with 50 μg/mL mouse secondary antibody (R&D Systems, catalog no. AF007) for 20 minutes at 37°C. Live cell imaging of membrane tension and intracellular Ca2+ during ICAM-1 clustering were performed using a Leica SP8 or an Olympus IX81 microscope (see above). For immunoblot analysis, beads were isolated using a magnetic holder (Miltenyi Biotec), and cells were lysed with RIPA buffer as described above.
Bbig i1
Manufactured by R&D Systems
Sourced in Japan
The BBIG-I1 is a laboratory equipment product designed for scientific research applications. It functions as a tool for analysis and data collection, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Details about the intended use or interpretation of the product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Bbig v1, by R&D Systems (2 mentions)
Magnetic holder, by Miltenyi Biotec (1 mentions)
Af007, by R&D Systems (1 mentions)
M 280, by Thermo Fisher Scientific (1 mentions)
Mab002, by R&D Systems (1 mentions)
Alexa fluor conjugates, by Thermo Fisher Scientific (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Rabbit anti human ve cadherin, by Cell Signaling Technology (1 mentions)
Fv1200, by Olympus (1 mentions)
Stage top incubator, by Tokai Hit (1 mentions)
5 protocols using bbig i1
1
Investigating ICAM-1 Clustering Dynamics
2
NK Cell Adhesion and Cytoskeletal Analysis
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MatTek dishes were coated overnight at 4°C with ICAM-1 (12.5 μg/mL with 100 μL per 10-mm well). NK cells were treated with 10 μM PMA (phorbol 12-myristate 13-acetate) for 30 min, added to ICAM-1-coated MatTek dishes (1 x 105 cells per well) and incubated for 1 hr at 37°C to allow cells to attach to the glass surface. Cells were fixed and permeabilized in one step, using 4% PFA (paraformaldehye) with 0.3% Triton X-100 in PBS for 10 min. After being washed with PBS, cells were blocked with 5% BSA in PBS and then incubated with primary and secondary antibodies diluted in 3% BSA in PBS. ProLong Gold (Invitrogen, Carlsbad, CA) was used as the mounting agent.
Primary antibodies included rabbit anti-human HS1 (D83A8), rabbit anti-human phospho-Tyr397 HS1, rabbit anti-human VE-cadherin (all from Cell Signaling Technology, Danvers, MA) and mouse anti-human ICAM-1/CD54 (monoclonal antibody BBIG-I1, R&D Systems). Secondary antibodies were Alexa-fluor conjugates (Invitrogen). Alexa-fluor conjugated phalloidin was used for F-actin.
Primary antibodies included rabbit anti-human HS1 (D83A8), rabbit anti-human phospho-Tyr397 HS1, rabbit anti-human VE-cadherin (all from Cell Signaling Technology, Danvers, MA) and mouse anti-human ICAM-1/CD54 (monoclonal antibody BBIG-I1, R&D Systems). Secondary antibodies were Alexa-fluor conjugates (Invitrogen). Alexa-fluor conjugated phalloidin was used for F-actin.
3
Melanoma Cell Transmigration Assay
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Hydrogels on coverslips were prepared with polyacrylamide as described [18] . The hydrogels were coated with fibronectin (10 µg/mL in PBS) by overnight incubation at 4°C and washed with PBS. HDMVECs were plated on the fibronectin-coated hydrogel substrates and incubated overnight to allow formation of monolayers. Monolayers were inspected by phase-contrast microscopy to ensure that ECs covered the substrate completely, without defects, before transmigration assays were performed. For blocking experiments, mouse monoclonal antibodies against ICAM1 (BBIG-I1, R&D Systems) or VCAM (BBIG-V1, R&D Systems) were diluted into EGM-2 MV media and added to monolayers 1 hr before assaying TEM.
Melanoma cells were added to HDMVEC monolayers in EGM-2 MV culture medium and placed into an environmental chamber (Stage Top Incubator, Tokai Hit, Shizuoka-ken, Japan) with 5% CO2 at 37°C on either an inverted wide-field microscope (Olympus IX72) or an inverted laser-scanning confocal microscope (Olympus FV1200). Wide-field fluorescence and DIC images were captured at 12-s intervals with a 10× or a 40× objective. Confocal fluorescence and DIC images were captured at 20-s intervals with a 60× objective.
Melanoma cells were added to HDMVEC monolayers in EGM-2 MV culture medium and placed into an environmental chamber (Stage Top Incubator, Tokai Hit, Shizuoka-ken, Japan) with 5% CO2 at 37°C on either an inverted wide-field microscope (Olympus IX72) or an inverted laser-scanning confocal microscope (Olympus FV1200). Wide-field fluorescence and DIC images were captured at 12-s intervals with a 10× or a 40× objective. Confocal fluorescence and DIC images were captured at 20-s intervals with a 60× objective.
4
Blocking ITGB1 in Cholangiocytes
5
Quantitative Immunodetection with Monoclonal Antibodies
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The same static binding technique described above was applied with the addition of mAbs at 5 µg/ml to the IE suspension prior adding it to the plates. All the mAbs were commercially available; 15.2 (AbD serotec), My13 (Invitrogen), 8.4A6 (Sigma), BBIG-I1 (R&D systems).
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