Reagents for NPs (liposomes) were purchased from Avanti Polar Lipids (Alabaster, AL). Human erythrocyte superoxide dismutase (SOD) was purchased from Sigma. NHS-PEG4-Dibenzocyclooctyne (DBCO) was purchased from Click Chemistry Tools (Scottsdale, AZ). IgG was purchased from Fisher Scientific. Anti-CD31 was purchased from Biolegend. PerCP/Cy5.5-conjugated anti-CD45 antibody was purchased from BioLegend. Monoclonal antibodies – anti-mouse-VCAM-1 (clone MK2.7), anti-mouse-ICAM-1 (clone YN1/1.7.4), and anti-mouse-PECAM-1 (MEC 13.3, Biolegend) – were produced by culturing hybridoma cells, purified using protein G sepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA) and dialyzed in PBS.
Percp cy5.5 conjugated anti cd45 antibody
Manufactured by BioLegend
Sourced in Japan
The PerCP/Cy5.5-conjugated anti-CD45 antibody is a fluorochrome-conjugated monoclonal antibody that binds to the CD45 antigen. CD45 is a transmembrane glycoprotein expressed on the surface of most hematopoietic cells. The PerCP/Cy5.5 fluorochrome is used to label the antibody, allowing for its detection and quantification in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Protein g sepharose, by GE Healthcare (2 mentions)
Human erythrocyte superoxide dismutase sod, by Merck Group (2 mentions)
Nhs peg4 dibenzocyclooctyne, by Vector Laboratories (2 mentions)
Anti mouse pecam 1 mec 13, by BioLegend (2 mentions)
Anti cd31, by BioLegend (2 mentions)
Collagenase type 4, by Worthington (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Worthington (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest pro software version 6, by BD (1 mentions)
4 protocols using percp cy5.5 conjugated anti cd45 antibody
1
Preparation and Characterization of NPs
2
Isolation and Culture of Murine Synovial Fibroblasts
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SFs were isolated from the paws of mice after one/two MSU-or zymosan-injections, and from noninjected naïve paws. SF cell suspensions of murine paws were prepared by gently rocking dissected and skin-flayed paws for 1 hour at 37°C in DMEM containing 1 mg/ml collagenase type IV (Worthington Biochemicals, #LS004188). Cells derived from 2 to 3 paws were pooled and cells were then grown in DMEM (Gibco) supplemented with 10% heat-inactivated calf serum, (glucose (4.5 g/L), L-glutamine 2 mM), penicillin and streptomycin (1% each) antibiotics, and 1% fungizone (Amphotericin B, Sigma Aldrich). Medium was changed twice a week. SFs between passages 4 and 8 were used for the experiments and contained less than 2% CD45 + or CD31 + cells, as determined by flow cytometry with a PerCP Cy5.5 conjugated anti-CD45 antibody (Biolegend, #103132) and an AF647-conjugated anti-CD31 antibody (Biolegend, #102516). Alternatively, we used flow cytometry sorted CD45 -CD31 -SFs.
3
Quantifying CTLs Against Tumor Antigens
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Since TC-1 cells express the HPV E7 tumor antigen [31 (link)], cytotoxic T cells (CTLs) against TC-1 were analyzed by counting E7-specific CTLs, and CTLs against T-01 were measured by a tetramer assay using glycoprotein B (gB), one of the HSV envelope proteins, 13 days after T-01 inoculation. Cells isolated from tumor tissues were suspended in staining buffer (RPMI 1640 with 200 U/mL penicillin, 0.2 mg/mL streptomycin, and 3% FBS) at a concentration of 1 × 106 cells/100 μL, and stained with phycoerythrin (PE)-labeled HPV16 E7 tetramer (MBL International Corporation, Nagoya, Japan) or PE-labeled HSV gB tetramer (MBL International Corporation), allophycocyanin (APC) labeled anti-CD3 antibody (clone: 17A2, Bio Legend Inc.), fluorescein isothiocyanate (FITC) labeled anti-CD8 antibody (clone: 53–6.7, Bio Legend), and peridinin-chlorophyll protein complex with cyanin-5.5 (PerCP-Cy5.5)-conjugated anti-CD45 antibody (clone: I3/2.3, Bio Legend Inc.) for 30 min in the dark. The cells were washed with staining buffer, measured with a FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA), and analyzed with Cell Quest Pro software version 6.0 (BD Biosciences). All flow cytometer measurements were performed under the same conditions.
4
Nanoparticle Fabrication and Antibody Conjugation
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Reagents for NPs (liposomes) were purchased from Avanti Polar Lipids (Alabaster, AL). Human erythrocyte superoxide dismutase (SOD) was purchased from Sigma. NHS-PEG4-Dibenzocyclooctyne (DBCO) was purchased from Click Chemistry Tools (Scottsdale, AZ). IgG was purchased from Fisher Scientific. Anti-CD31 was purchased from Biolegend. PerCP/Cy5.5-conjugated anti-CD45 antibody was purchased from BioLegend. Monoclonal antibodies -anti-mouse-VCAM-1 (clone MK2.7), anti-mouse-ICAM-1 (clone YN1/1.7.4), and antimouse-PECAM-1 (MEC 13.3, Biolegend) -were produced by culturing hybridoma cells, purified using protein G sepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA) and dialyzed in PBS.
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