Rat peripheral blood leukocytes were isolated from heparinized blood by red blood cell lysis. hSIRPA was detected using an anti-human SIRPA monoclonal antibodies (clones REA144, Miltenyi or SE5A5, BD Biosciences, both IgG1). Recombinant human CD47-Fc (hCD47-Fc, R&D Systems) was also used to detect human SIRPA expression since it does not bind to rat SIRPA. Rat SIRPA was detected using anti-rat SIRPA-FITC monoclonal antibody (OX41, European Collection of Cell Culture). Several isotype negative controls were used: APC-mouse IgG1, FITC-mouse IgG2a, PE-mouse IgG1 (BD Pharmingen). All monoclonal antibodies, hCD47-Fc and human IgGs were used at 10 ug/ml. Fluorescence was analyzed with a FACSVerse flow cytometer (BD Biosciences), and FlowJo software was used to analyze data.
Fitc mouse igg2a
Manufactured by BD
Sourced in United States
The FITC Mouse IgG2a is a fluorescently labeled immunoglobulin G (IgG) antibody. It is designed for use in various immunoassays and flow cytometry applications.
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12 protocols using fitc mouse igg2a
1
Isolation and Characterization of Rat Leukocytes
2
Multicolor Flow Cytometry Immunophenotyping
3
CD24 and CD44 Expression Analysis
4
Quantifying MRP1 Transporter Expression
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Expression levels of the MRP1 transporter in monolayers and dissociated spheroid cells were estimated using flow cytometry with FITC-labeled mouse anti-human MRP1 antibody QCRL-3 and an isotype control (FITC Mouse IgG2a) (BD Biosciences, cat. no. 557593 and 555573, respectively) according to the previously published procedure (22 (link)). Briefly, monolayers and spheroids were trypsinized with 0.25% trypsin/0.02% EDTA to form single cell suspensions, permeabilized with 80 µg/ml saponin, treated with the antibodies, and analyzed with a BD FACSCanto II flow cytometer.
5
Comprehensive Immune Cell Profiling
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FITC mouse anti‐CD1a (HI149), FITC mouse anti‐CD11b (LT11), FITC mouse anti CD11c (BU15), FITC mouse anti‐CD31 (MEM‐05), FITC mouse anti‐CD33 (HIM3‐4), FITC mouse anti‐CD40 (HI40a), FITC mouse anti‐CD44 (MEM‐85), FITC mouse anti‐CD54 (1H4), FITC mouse anti‐CD80 (MEM‐233), FITC mouse anti‐CD86 (BU63; ImmunoTools), FITC mouse anti‐CD14 (M5E2), FITC mouse anti‐HLA‐DR (G46‐6), phycoerythrin (PE) mouse anti‐CD68 (Y1/82A; BD Bioscience), allophycocyanin (APC) mouse anti‐CD163 (GHI/61), APC mouse anti‐CD206 (15‐2), APC mouse anti‐PDL1 (29E.2A3; BioLegend, San Diego, CA, USA) and isotype controls FITC mouse IgG1 (PPV‐06), FITC mouse IgG2b (PLRV219; ImmunoTools), FITC mouse IgG2a (G155‐178), PE mouse IgG2b (27‐35; BD BioScience), APC mouse IgG2b (MPC‐11; BioLegend) were used for flow cytometry.
Primary mouse anti‐CD30 (Ber‐H2) and mouse anti‐CD68 (KP1), anti‐Prox1, anti‐CD206 (D‐1; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and secondary goat anti‐mouse horseradish peroxidase (HRP) polyclonal; Agilent, Santa Clara, CA, USA) were used for peroxidase and immunofluorescence staining.
ELISA kits for the detection of secreted M‐CSF and IL‐13 in cell culture supernatants were purchased from R&D Systems (Minneapolis, MN, USA). The sensitivity was 11.2 and 13.2 pg·mL−1, respectively.
Primary mouse anti‐CD30 (Ber‐H2) and mouse anti‐CD68 (KP1), anti‐Prox1, anti‐CD206 (D‐1; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and secondary goat anti‐mouse horseradish peroxidase (HRP) polyclonal; Agilent, Santa Clara, CA, USA) were used for peroxidase and immunofluorescence staining.
ELISA kits for the detection of secreted M‐CSF and IL‐13 in cell culture supernatants were purchased from R&D Systems (Minneapolis, MN, USA). The sensitivity was 11.2 and 13.2 pg·mL−1, respectively.
6
Flow Cytometry Antibody Labeling
7
Multicolor Flow Cytometry Immunophenotyping
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PE mouse IgG1(BD), APC Rat IgG2a (BD), PerCp/Cy5.5 mouse IgG2b (Biolegend), Alexa Fluor 700 Mouse IgG1 (BD), PE-Cy7 Mouse IgG2a (BD), V450 Mouse IgG1 (BD), APC Cy7 Mouse IgG1 (Biolegend), FITC Mouse IgG2a (BD).
Following staining, cells were fixed with 2% paraformaldehyde and run on a BD Biosciences LSR II flow cytometer (San Jose, CA, USA). Data were analyzed using Winlist 6.0 (Topsham, ME, USA). Results are reported as the percent of the phenotype being analyzed in the total peripheral blood mononuclear cell pool.
Following staining, cells were fixed with 2% paraformaldehyde and run on a BD Biosciences LSR II flow cytometer (San Jose, CA, USA). Data were analyzed using Winlist 6.0 (Topsham, ME, USA). Results are reported as the percent of the phenotype being analyzed in the total peripheral blood mononuclear cell pool.
8
Antibody Detection and Cytotoxicity Assays
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The following antibodies were used: Alexa Fluor 647–coupled anti-GFP (catalog no. 565197, BD Pharmingen); Alexa Fluor 647 rat immunoglobulin G2a (IgG2a), κ isotype control (catalog no. 400526); Alexa Fluor 647 anti-human CD195 (catalog no. 313712) from BioLegend; phycoerythrin (PE) mouse IgG1, κ isotype control (catalog no. 550617); PE mouse IgG2a, κ isotype control (catalog no. 553457); fluorescein isothiocyanate (FITC) mouse IgG2a, κ isotype control (catalog no. 555573); FITC mouse IgG1 κ isotype control (catalog no. 555748); PE mouse anti-human CD184 (catalog no. 555974); FITC mouse anti-human CD4 (catalog no. 555346); FITC mouse anti-human CD3 (catalog no. 555332); and PE mouse anti-human CD11b/Mac1 (catalog no. 555388) from BD Biosciences.
The amount of lactate dehydrogenase (LDH) released into supernatants was quantified using a Pierce LDH Cytotoxicity Assay kit (catalog no. 88953, Thermo Fisher Scientific). The Alliance HIV-1 p24 ELISA (enzyme-linked immunosorbent assay) Kit (catalog no. NEK050, PerkinElmer) was used to determine the amount of capsid p24 protein in supernatants and cell lysates. LDH and p24 quantification were performed following the manufacturer’s instructions.
The amount of lactate dehydrogenase (LDH) released into supernatants was quantified using a Pierce LDH Cytotoxicity Assay kit (catalog no. 88953, Thermo Fisher Scientific). The Alliance HIV-1 p24 ELISA (enzyme-linked immunosorbent assay) Kit (catalog no. NEK050, PerkinElmer) was used to determine the amount of capsid p24 protein in supernatants and cell lysates. LDH and p24 quantification were performed following the manufacturer’s instructions.
9
Detailed Immunoblotting and Cell Phenotyping
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Unless otherwise specified, chemicals and reagents were purchased from Sigma Aldrich. Antibodies against: p53 (DO-1) and p21Cip1 (C-19) were purchased from Santa Cruz Biotechnology, Ki-67 from Dako, PARP-1 from Enzo, p-p53 (Ser15), p-S6 (Ser235/236), and Nanog from Cell Signaling, p62 from Transduction LaboratoriesTM, γ-H2A.X (Ser 139) from Abcam, H2A.X and GAPDH from Millipore. Secondary anti-mouse and anti-rabbit antibodies conjugated with HRP were obtained from Vector Laboratories, and ECL reagents from Thermo Scientific. LysoTracker® Red DND-99, DilC12(3) (Dil), secondary antibodies conjugated with AlexaFluor 488 or AlexaFluor 555 were purchased from Molecular Probes. Mounting medium, protease inhibitor cocktail and phosphatase inhibitor cocktail were obtained from Roche Diagnostics. 7-AAD, Matrigel Matrix, Matrigel Matrix Growth Factor Reduced, FITC mouse anti-human CD24, FITC mouse IgG2a, κ isotype control, AlexaFluor® 700 mouse IgG2b, κ isotype control, Alexa Fluor®700 mouse anti-human CD44 were obtained from BD PharmingenTM, APC mouse IgG1 isotype control, APC mouse anti-human CD133/1 (AC133) were purchased from Miltenyi Biotec. ELISA kits for human vascular endothelial growth factor (VEGF), and human interleukin-8 (IL-8), neutralizing antibodies against human VEGF and corresponding isotype control antibodies were procured from R&D Systems.
10
Flow Cytometry Analysis of SRDC and PECs
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SRDC and PECs were stimulated as described above (2.5.). Supernatant was centrifuged at 300×g to pool floating cells and attached cells after their detachment using accutase (Affymetrix eBioscience). After centrifugation at 300×g, supernatant was removed for quantification of cytokines (section 2.5 .) and pelleted SRDC were suspended and saturated for 30 min on ice in PBS containing 1% bovine serum albumin, 2% mouse serum and 0.1% azide (PBS-BSA-azide). After centrifugation, 3 × 105 SRDC were incubated for 30 min on ice in the dark in PBS-BSA-azide with 0.5 μg fluorescein isothiocyanate (FITC) mouse anti-mouse H–2K[k] MHC class I antibody (clone 36-7-5, BD Pharmingen™), 0.5 μg FITC mouse anti-mouse I-E[k] MHC class II antibody (clone 14-4-4S, BD Pharmingen™), or 0.5 μg FITC mouse IgG2a, κ isotype control (clone G155-178, BD Pharmingen™). After centrifugation, SRDC were suspended in 300 μL PBS-2% paraformaldehyde and analysed by flow cytometry (BD FACSCalibur™ and CellQuest™ software, BD Bioscience). PECs were incubated for 15 min at 4 °C in the dark in 100 μL binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 5 mM CaCl2) with 5 μL annexin V-FITC (BD Pharmingen™) and 5 μg/mL propidium iodide (Sigma). After centrifugation, PECs were suspended in 300 μL binding buffer and analysed by flow cytometry.
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