All antibody staining was performed in HBSS buffer (Corning #21021CV) containing Pen/Strep (100 Units/mL; Fisher Scientific #MT30002CI), HEPES (10uM; Life Technologies #15630080) and SerumPlus II Medium Supplement (2%; Sigma #14009C). Bone marrow cells isolated from tibias, femurs, and iliac crests were combined for calculating total BM from each mouse. Peripheral blood, bone marrow, and spleen cells were suspended in complete HBSS (1.0×108 cells/mL) and incubated on ice for >20 minutes with the following antibodies as needed (all 1:100 dilution): anti-mouse CD45.2-BV421 (BioLegend #109831), anti-mouse Ly-6A/E (Sca-1)-APC (BioLegend # 122512), anti-mouse c-Kit-BV421 (BioLegend #105828), anti-mouse CD25-PE (BD Biosciences #553866), anti-mouse CD3e-APCcy7 (BioLegend #100330), anti-mouse CD4-APC (BioLegend #100515), anti-mouse CD8-PEcy7 (BD Biosciences #552877), anti-human CD45-APC (BioLegend #368512), anti-human CD7-FITC (BioLegend #343104).
Apc anti human cd45
Manufactured by BioLegend
Sourced in United States
APC anti-human CD45 is a fluorescently-labeled antibody that binds to the CD45 cell surface antigen, which is expressed on most human hematopoietic cells. This product is intended for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti mouse cd45, by BioLegend (3 mentions)
7 aad, by BioLegend (2 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Apc anti mouse cd4, by BioLegend (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Serum plus 2 medium supplement, by Merck Group (1 mentions)
Anti mouse cd8 pecy7, by BD (1 mentions)
Anti mouse cd45.2 bv421, by BioLegend (1 mentions)
Facs lysing solution, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Apc anti mouse cd45, by BioLegend (1 mentions)
Fitc anti human cd8, by BioLegend (1 mentions)
Pe cyanine7 anti human cd4, by BioLegend (1 mentions)
Apc anti human cd4, by BioLegend (1 mentions)
Pe anti mouse f4 80, by BioLegend (1 mentions)
Pe anti mouse human cd11b, by BioLegend (1 mentions)
Anti mouse f4 80 pe cyanine 7, by BioLegend (1 mentions)
Anti mouse ly6g fitc, by BioLegend (1 mentions)
10 protocols using apc anti human cd45
1
Multiparameter Flow Cytometry Immunophenotyping
2
Isolation and Intracellular Staining of Mouse PBMCs
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PBMCs were isolated from mouse peripheral blood using BD FACS Lysing Solution (BD Biosciences, CA, USA) according to the manufacturer’s protocol. Cell pellets were re-suspended in 2 ml of 1X lysing solution and incubated at room temperature for 15 minutes. After lysis, samples were pelleted at 700 g in a microcentrifuge for 5 min at room temperature, and stained with 100 μl monoclonal antibody mixture containing 2 μl of each anti-human CD45-APC and CD14-Percyp-cy5.5 (Biolegend, CA, USA). The monocyte labeling was performed for 60 min at 4°C. Before intracellular staining, cell pellets were washed with 1 ml phosphate buffered saline (PBS) supplemented with 2% fetal bovine serum and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Kit. The fixed/permeabilized cells were thoroughly resuspended in 50 μl of BD Perm/Wash buffer containing a pre-determined optimal concentration of a Gn-specific camel antibody and subsequently stained with FITC-conjugated anti-camel IgG Fc. Pelleted cells were resuspended in 200 μl of wash buffer and analyzed on flow cytometer (NovoCyte Flow Cytometer, ACEA). Cells were firstly gated for human CD45 before analyzing other parameters.
3
Multiparametric Flow Cytometry for Tumor Immune Profiling
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For blood and tumor samples from mice, the following antibodies were purchased from BioLegend: anti-human CD45-APC (BioLegend Cat# 304011, RRID:AB_314399), anti-human CD45-Brilliant Violet 421™ (BioLegend Cat# 304032, RRID:AB_2561357), anti-human CD4-APC (BioLegend Cat# 300514, RRID:AB_314082), anti-human CD4-PE/Cyanine7 (BioLegend Cat# 300512, RRID:AB_314080), anti-human CD8-FITC (BioLegend Cat# 980908, RRID:AB_2888883), anti-human CD8-Brilliant Violet785™ (BioLegend Cat# 344739, RRID:AB_2566201), anti-mouse CD45-APC (BioLegend Cat# 103111, RRID:AB_312976), anti-mouse CD45-Brilliant Violet 711™ (BioLegend Cat# 103147, RRID:AB_2564383), anti-mouse/human CD11b-Brilliant Violet 570™ (BioLegend Cat# 101233, RRID:AB_10896949), anti-mouse/human CD11b-PE (BioLegend Cat# 101208, RRID:AB_312791), anti-mouse/human CD11b-PE/Cyanine7 (BioLegend Cat# 101215, RRID:AB_312798), anti-mouse Ly6G-FITC (BioLegend Cat# 127606, RRID:AB_1236494), anti-mouse Ly6G-PerCP/Cyanine5.5 (BioLegend Cat# 127616, RRID:AB_1877271), anti-mouse Ly-6C-PerCP/Cyanine5.5 (BioLegend Cat# 128012, RRID:AB_1659241), anti-mouse F4/80-PE/Cyanine7 (BioLegend Cat# 123114, RRID:AB_893478), anti-mouse F4/80-PE (BioLegend Cat# 123110, RRID:AB_893486).
4
Engraftment of Myelodysplastic Stem Cells in Mice
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HSPCs (CD34+) derived from individuals with LR-MDS and HR-MDS were transfected with 20 nM of SCR or mTOG-Ψ RNA oligos. The cells were harvested 6 h post transfection and 1 × 105 cells were injected into sub-lethally irradiated (250 cGy) NSG-S mice (11–14 weeks old) via the tail vein. Human engraftment was followed in the peripheral blood of the transplanted mice every 2 weeks. After 8 weeks, the mice were killed and their BM cells were harvested, treated with ammonium chloride solution (StemCell Technologies) to lyse the red blood cells, and stained. The following antibodies were used: anti-mouse CD45–Alexa Fluor 700 (1:200; BioLegend), anti-human CD45–APC (1:200; BioLegend), anti-human CD19–BV605 (1:200; BD Biosciences), anti-human CD15–PE (1:200; BioLegend), anti-human CD33–PE (1:200; BD Biosciences), anti-human CD34–BV421 (1:100; BioLegend), anti-human CD123–BV605 (1:50; BioLegend) and anti-human CD45RA–FITC (1:200; Thermo Scientific). The cells were then washed, resuspended in 1 μg ml−1 7-AAD (BioLegend) in PBS + 3% FBS and analysed using an LSRFortessa X-20 flow cytometer (BD Biosciences).
5
Humanized Mouse Model for Hematopoietic Engraftment
6
Quantifying Macrophage GSDMD Expression
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The number of macrophages and the relative expression of GSDMD were determined by flow cytometry. In brief, follicular fluids without obvious blood stain were collected from discarded clinical specimens and then centrifuged at 500 g for 5 mins at 4 °C. Cells were washed two times with ice‐cold PBS and blocked in 2% BSA before staining with antibodies. To avoid non‐specific staining, the anti‐GSDMDC1 antibodies (NBP2‐80427, NOVUS) were first labelled with FITC using Antibody Labeling Kit. Other antibodies included APC anti‐human CD45 (368 511, Biolegend) and PE anti‐human CD14 antibodies (301 805, Biolegend). One blank tube and 2 single‐dye adjustment compensation tubes were used as controls. Single cells were first gated using FSC‐A/SSC‐A, followed by FSC‐H/FSC‐A to remove doublet. Then gate was set on CD14+ CD45+ cells, and the MFI of GSDMD was determined by flow cytometry. Assays were performed on CytoFLEX Flow Cytometer (Beckman, Inc) and analyzed by FlowJo (10.8.1)
7
Immunophenotyping of Glioma Cells
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The PD-L1 expression was determined by FACS using a PD-L1 specific (Thermo Fisher, #53-5983-42) or a mouse IgG1κ isotype control antibody (Thermo Fisher, #53-4714-80). For the surface exposure of calreticulin (CRT), infected glioma cells at 50% oncolysis were collected and stained with a CRT specific antibody (Novus Bio #B-4-120621). For human immune cell engraftment in mice, blood was taken, mixed 1:1 with 10 μM EDTA, and incubated with both the FITC-anti-mouse CD45 (Biolegend, Eching, Germany, #103108) and APC-anti-human CD45 (Biolegend, #304012) antibodies. After washing, the erythrocytes were removed by ACK buffer lysis (Thermo Fisher). Analyses were performed on a MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec, Bergisch-Gladbach, Germany), and data were analyzed using FlowJo v10 (Ashland, OR, USA).
8
Immune Cell Phenotyping by Flow Cytometry
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PE/Cyanine anti‐human CXCR5 and APC anti‐human CD45 antibodies were purchased from Biolegend (San Diego, USA). Cells were stained for flow cytometry (Beckman Coulter) and then analyzed by Kaluza analysis software.
9
Quantification of Human Hematopoietic Cells
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A Miltenyi MacsQuant 10 instrument was used for flow cytometric analysis of bone marrow hematopoietic cell populations. To assess for human hematopoietic stem and progenitor cell populations the following antibodies purchased from Biolegend were used: FITC antimouse CD45 (103108), APC antihuman CD45 (368512), Pacific Blue antihuman Lineage Cocktail (348805; CD3, CD14, CD16, CD19, CD20, CD56), PE antihuman CD34 (343506), Brilliant Violet 510 antihuman CD38 (356612), and 7-AAD Viability Staining Solution (420404). Human lineage analysis was performed using the 7-color immunophenotyping kit (130–098-456; Miltenyi Biotec).
10
Human Hematopoiesis Reconstitution in NSG Mice
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Human engraftment in NSG mice was determined 2 months post-transplantation from peripheral blood using a fluorescein isothiocyanate (FITC) anti-human CD45 (BioLegend, San Diego, CA, USA) and a phycoerythrin (PE) anti-mouse CD45 (BioLegend, San Diego, CA, USA). Hematopoiesis reconstitution was determined at the time of sacrifice, 12–16 weeks post-transplantation, from peripheral blood using the following conjugated antibodies: allophycocyanin (APC)/Cyanine7 anti-human CD19, FITC anti-human CD3, PE anti-human CD33, and APC anti-human CD45 (BioLegend, San Diego, CA, USA). All of the flow cytometric analyses were performed using the BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA).
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