To block the binding of ICAM-1 to LFA-1, activated T cells were added to the cultures of glia either after 1 hour of preincubation of glia with 10 μg/mL anti–ICAM-1 (BD Pharmingen) or isotype antibody (IgG1κ, Biolegend) or after 30 minutes of preincubation of T cells as well as glia with 0.1 mmol/L LG (SAR-1180, Sigma-Aldrich) dissolved in dimethyl sulfoxide and H2O (1:1 v/v).
Igg1κ
Manufactured by BioLegend
Sourced in United States
IgG1κ is an antibody isotype that serves as a common control or reference standard in various immunological assays. It binds to the Fc region of IgG1 antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Anti icam 1, by BD (1 mentions)
Lsm 710 confocal, by Leica camera (1 mentions)
Dmlb fluorescent microscope, by Leica camera (1 mentions)
α sma ab5694, by Abcam (1 mentions)
Anti human cd49d, by BioLegend (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Anti human cd19, by BioLegend (1 mentions)
Anti human cd29, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Flow cytometry staining buffer, by BioLegend (1 mentions)
Fixation and permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Cd56 bv421, by BD (1 mentions)
Cd3 apc cy7, by BD (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Igg2aκ, by Thermo Fisher Scientific (1 mentions)
6 protocols using igg1κ
1
Inhibition of ICAM-1-LFA-1 Binding
2
Multicolor Immunofluorescence Staining of Tissue
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Frozen samples, processed immediately after tissue harvesting, were cut in a Leica CM1900 cryostat. For staining, tissues were fixed in ice-cold acetone:methanol (50:50) and antibodies were prepared in antibody diluent (003118; Invitrogen-Thermo Fisher Scientific, Paisley, UK). After staining, slides were mounted in Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI;F6057; Sigma, St Louis, MO, USA). Primary antibodies used were CD146-FITC (MCA2141F; AbD Serotec-BioRad, Kidlington, UK), CD144 (AHP628Z; AbD Serotec-BioRad), NG2 (MAB2585; R&D Systems, Minneapolis, MN, USA), αSMA (ab5694; AbCam, Cambridge, UK), CD29 (303015; BioLegend, San Diego, CA, USA), CD44 (AbD Serotec-BioRad), and CD34 (21270341S; ImmunoTools, Friesoythe, Germany). Isotype controls were IgG1-FITC (MCA928F; AbD Serotec-BioRad), IgG1 (MAB002; R&D Systems), IgG1κ (400101; BioLegend), and IgG1 (21335011; ImmunoTools), all raised in mouse; and rabbit IgG (PRABP01; AbD Serotec-BioRad). Secondary antibodies were conjugated to AF488 (A11029), AF568 (A110037), and AF568 (A10042), all from Invitrogen-Thermo Fisher Scientific. Micrographs were produced using a Zeiss LSM710 confocal or Leica DMLB fluorescent microscope.
3
Flow Cytometry Analysis of B-ALL and HUVEC
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After harvesting, B-ALL cells or HUVECs (0.1-1x106) were centrifuged at 500 g for 5 min at room temperature and resuspended in 100 µl PBS containing anti-human CD19 (#302206, 1:20 dilution, Biolegend, Inc.), anti-human CD49d (#304304, 1:20 dilution, Biolegend, Inc.) and anti-human CD29 (#303008, 1:20 dilution, Biolegend, Inc.) antibodies, alongside the isotype control mouse IgG1κ (Biolegend, Inc.). Following incubation at 4˚C for 30 min, cells were washed with 1 ml PBS and resuspended in 100 µl DAPI/PBS (0.1µg/ml) for 15 min at 4˚C for assessment using a flow cytometer (BD FACSCanto II, BD Biosciences). Flow cytometry data were analyzed from the viable cells in the DAPI-negative population, based on an isotype gating strategy using Flow Jo (version 10, BD Biosciences).
For apoptosis assay, B-ALL cells were treated for 48 h with either DMSO or AVA4746 25 µM, with or without VDL chemotherapy (vincristine 5 nM, dexamethasone 0.05 nM, L-asparaginase 2.5x10-3 IU/ml) in the presence or absence of OP9. Annexin V (#640947, 1:20; Biolegend, Inc.) and DAPI were used to stain the 1x106 cells for 15 min at room temperature (25˚C), in the dark.
For apoptosis assay, B-ALL cells were treated for 48 h with either DMSO or AVA4746 25 µM, with or without VDL chemotherapy (vincristine 5 nM, dexamethasone 0.05 nM, L-asparaginase 2.5x10-3 IU/ml) in the presence or absence of OP9. Annexin V (#640947, 1:20; Biolegend, Inc.) and DAPI were used to stain the 1x106 cells for 15 min at room temperature (25˚C), in the dark.
4
Immunophenotyping of peripheral blood mononuclear cells
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Peripheral blood mononuclear cells were isolated from fresh blood and resuspended at 10 × 106/ml in flow cytometry staining buffer (#420201, BioLegend). Then, cells were incubated with fluorescent conjugated antibodies, including APC‐Cy7‐CD3 (#557757, BD Pharmingen) and BV421‐CD56 (#562751, BD Pharmingen), on ice for 20 min in the dark. To detect the expression of intracellular cytotoxic molecules, cells were fixed and permeabilized using Fixation and Permeabilization Buffer Set (#88‐8824, eBioscience), and then stained with GZMB antibody (#515406, BioLegend) or IgG1 κ (#400136, BioLegend) as an isotype control. After washing twice by centrifugation at 350 g for 5 min, cells were resuspended in 0.5 ml of staining buffer and harvested using CytoFlex S (Beckman Coulter). Data analysis was performed using FlowJo software (version 10).
5
Blocking TIL-Melanoma Interactions
6
PMN Adhesion to hCMEC/D3 Monolayers
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PMN (2 × 107/mL) suspended in PBS were stimulated with CCM or SSCM at 23°C for 10 minutes in a total volume of 0.2 mL. Subsequently, function neutralizing monoclonal antibodies (all IgG1κ subclass; BioLegend, San Diego, CA, USA) directed against either human β2-integrin (clone TS 1/18; 25 μg/mL), human LFA-1 (clone HI111; 5 μg/mL), or Mac-1 (clone ICRF44; 5 μg/mL), were added to the PMN suspension for an additional 15 minutes. An isotype control (IgG1κ; BioLegend, San Diego, CA, USA) antibody was used in parallel experiments at the corresponding concentrations. Following treatment, PMN were immediately added to VascuLife EnGS-Mv cell culture medium to a final concentration of 1 × 106/mL and perfused over hCMEC/D3.
In some experiments, hCMEC/D3 grown in laminar flow micro channels (ibidi GmbH) were stimulated with either CCM or SSCM and subsequently treated with function neutralizing anti-human ICAM-1 (CD54; clone HCD54; 5 μg/mL) or isotype control (both IgG1κ subclass; BioLegend, San Diego, CA, USA) antibodies in VascuLife EnGS-Mv cell culture medium for 30 minutes at 37°C, preceding PMN perfusion.
In some experiments, hCMEC/D3 grown in laminar flow micro channels (ibidi GmbH) were stimulated with either CCM or SSCM and subsequently treated with function neutralizing anti-human ICAM-1 (CD54; clone HCD54; 5 μg/mL) or isotype control (both IgG1κ subclass; BioLegend, San Diego, CA, USA) antibodies in VascuLife EnGS-Mv cell culture medium for 30 minutes at 37°C, preceding PMN perfusion.
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