Cells isolated from humanized mice were prepared for flow cytometry as described above and stained with the following extracellular markers: anti-mouse CD45-BV605 (BD), anti-human CD45-APC (BD), anti-human CD3-APC-eFluor780 (Thermo Fisher), anti-human CD8-AF700 (BD), and anti-human CD33-BV711 (BD). Cells were fixed with Cytofix buffer (BD) at 37°C for 10 min. Following this, cells were permeabilized with permeabilization buffer III (BD) for 30 min at 4°C and stained with the following phosflow antibodies: anti-human pSTAT1-BV421 (BD), anti-human pp65-PerCP-eFluor710 (Life Technologies), anti-human pIRF3 S396-PE (Cell Signaling, MA, USA), and anti-human pTBK-1 (Cell Signaling).
Anti human cd45 apc
Manufactured by BD
Sourced in United States
Anti-human CD45-APC is a monoclonal antibody conjugated with allophycocyanin (APC) that binds to the CD45 antigen expressed on human leukocytes. It is a useful tool for the identification and enumeration of leukocyte populations in flow cytometry analysis.
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Lab products found in correlation
Cd4 pe, by BD (2 mentions)
Anti mouse cd16 cd32, by BioLegend (2 mentions)
Anti human cd34 pe, by BioLegend (2 mentions)
Anti human cd11b pe, by BD (2 mentions)
Anti human cd19 pe cy7, by BioLegend (2 mentions)
Rbc lysis buffer, by Santa Cruz Biotechnology (2 mentions)
Anti mouse cd45 percp cy5, by BioLegend (2 mentions)
Anti human cd3 pe dazzle 594, by BioLegend (2 mentions)
Anti mouse cd45 percp, by BD (2 mentions)
Cytofix buffer, by BD (1 mentions)
Permeabilization buffer 3, by BD (1 mentions)
Cd90 percp cy5, by BD (1 mentions)
Anti mouse igg beads, by BD (1 mentions)
Facs fortessa, by BD (1 mentions)
Cd31 fitc, by BD (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Human serum, by Merck Group (1 mentions)
Kaluza software version 1, by Beckman Coulter (1 mentions)
Cd166 pe, by BD (1 mentions)
Abc anti mouse bead kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti human cd45 apc
1
Multiparametric Flow Cytometry Analysis
2
Adipogenic Potential Evaluation by Flow Cytometry
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Flow cytometry was performed on cells from passage three to evaluate adipogenic potential of the isolated cells. The cells were proliferated according to standard protocol in DMEM/F12 medium supplemented with 10% FBS, 1% PS, and 1 nmol/L fibroblast growth factor in 5% CO2, 37 °C environment until they reached 80% confluence. Cells were harvested using TrypLE (Gibco; Life Technologies) followed by a wash in buffer (PBS containing 2% FBS and 0.01% NaN3) and were afterwards resuspended in staining buffer (PBS containing 2% FBS, 1% Human Serum [catalog #1001291552; Sigma] and 0.01% NaN3). Anti-human CD45-APC, CD31-FITC, CD90- PerCP.Cy5.5, and CD166-PE (BD Pharmingen), antibodies were added to the cells and flow cytometry was applied for quantification using a FACS Fortessa (BD Bioscience). For compensation, single stain was used with one drop of negative control beads and anti-mouse IgG beads (BD Biosciences). Data analysis was performed using Kaluza software, version 1.2 (Beckman Coulter).
3
Multicolor Flow Cytometry Analysis
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Reconstitution with human immune cells in different mouse organs as well as activation of splenocyte-derived T cells and Jurkat cells was assessed by multicolor flow cytometry. The following monoclonal antibodies (mAbs) were used: anti-human CD45-APC (HI30), CD19-FITC (HIB19), CD3-APC-H7 (SK7), CD4-PE (SK3), CD8-FITC and CD8-PE (HIT8a), CD25-BV421 (M-A251), CD69-PE and CD69-FITC (FN50), Nkp46-PE-Cy7 (9E2/NKp46), CD56-PE (MY31), and IFN-ɣ-PerCP-Cy5.5 (B27) from BD Biosciences. To reduce nonspecific binding, human and/or mouse BD Fc Block were used (BD Biosciences). To determine the viability of cells, samples were incubated with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies) according to the manufacturer’s instructions. Dead cells were excluded from the analysis by gating out low forward scatter and brightly aqua fluorescent reactive dye-retaining cells. For compensations, single staining and/or AbC anti-mouse bead kit (Life Technologies) were used. To identify gating boundaries, unstained, single-stained, and/or fluorescence minus one (FMO) controls were used. Samples were acquired with a BD FACSCanto flow cytometer (BD Biosciences) at the VA hospital Flow Cytometry Core facility using established protocols. Data analysis was performed using FlowJo version 10 (FLOWJO) software.
4
Multiparameter Flow Cytometry of Hematopoietic Cells
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50 μL peripheral blood or ~5×105 bone marrow cells in PBS with 10% FBS were blocked with anti-mouse CD16/CD32 (cat # 101302, Biolegend, San Diego, CA, USA) and then stained with fluorophore conjugated antibodies. Following staining, red blood cells were lysed with RBC lysis buffer (cat # sc-296258, Santa Cruz Biotechnology, Dallas, TX, USA) for 10 min and washed twice with 10% FBS in PBS. Peripheral blood samples underwent a second 5 min RBC lysis prior to washing. Samples were acquired on a BD Accuri C6 or Beckman coulter Gallios flow cytometer and analyzed using BD Accuri Csampler software or FlowJo. Cells were stained with anti-human CD34 PE (cat # 343606, Biolegend), anti-human CD11b PE (cat # 555388, BD Biosciences, San Jose, CA, USA), anti-human CD3 PE/Dazzle™ 594 (cat # 300450, Biolegend), anti-mouse CD45 PerCP-Cy5.5 (cat # 103132 Biolegend), anti-mouse CD45 PerCP (cat # 557235, BD), anti-human CD19 PE-Cy7 (cat # 302216, Biolegend), anti-human CD45 APC (cat # 555485, BD Bioscience or cat # 304012, Biolegend). Analysis based 10 000 bone marrow and 3 000 blood cells from the live cell gate.
5
Characterization of Exosome Origins
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Anti-CD81 (sc-166029), anti-CD9 (sc-13118), anti-Hsp70 (sc-32239), and anti-hnRNPA2/B1 (sc-53531) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). From Abcam (Cambridge, MA, USA) were anti-caveolin-1 (Cav-1, ab2910) and anti-caveolin-3 (Cav-3, ab2912). The antibodies used in flow cytometry to characterize the origin of exosomes were anti-human CD142-PE to monocytes and endothelial cells (#550313), anti-human CD146-PE-CF594 to endothelial cells (#564327), anti-human CD45-APC (#555485) to leucocytes and hematopoietic cells, and anti-human CD36-PerCP-Cy (#561536) to platelets from BD Biosciences (San Jose, CA, USA). Hsp70 was used as a specific marker of cardiomyocyte-derived exosomes [46 (link)]. The Megamix-Plus FSC Beads were acquired from BioCytex (BioCytex, Marseille, France) and aldehyde/sulfate latex beads with 4 μm (A37304) were from Invitrogen™ (Waltham, MA, USA), other reagents used are mentioned where required.
6
Isolation and Characterization of Tumor and Immune Cells
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The orthotopic HCC tumor tissues or subcutaneous xenografts were dissected out and minced. Then tissues were digested with 0.8 mg/mL Collagenase IV (Sigma, USA) at 37 ˚C for 1 h. The cell suspensions were filtered through 70 μm strainer and resuspended in 36% Percoll (GE Healthcare, UK). PBMCs of anonymous human healthy donors were isolated by Ficoll reagent (Sigma, USA) according to manufacturers’ instructions. For cell surface staining, 1 × 106 cells were incubated with anti-Fc receptor blocking antibody (2.4G2) at 4 ˚C for 15 min. Murine samples were stained with anti-mouse CD45 APC, CD3 FITC, Gr-1 V450, CD4 PE, CD8a V450, CD25 APC-CY7, Ly6C FITC, Ly6G PECY7, CD11 PE, and F4/80 APC-CY7 from BD Bioscience (USA). Human samples were stained with anti-human CD45 APC, CD11 PE, CD33 FITC, and HLA-DR V450 from BD Bioscience (USA). For intracellular staining of Arg1, Foxp3, and Ki67, cells were fixed and permeabilized by Fixation/Permeabilization solution (BD Biosciences, USA) at 4 ˚C for 15 min. Then cells were washed and stained with anti-mouse Arg1, anti-mouse Foxp3, and anti-Ki67 from BD Bioscience (USA). Flow cytometry was performed on a B.D. Influx cell sorter (BD Bioscience, USA). Flowjo software was used to analyze the data.
7
Multiparameter Flow Cytometry Analysis of Engraftment Levels
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Cells were stained using the following antibodies: CD45‐Pacific blue (Biolegend #304029), GPR56‐PE (Biolegend #358204), CD11b‐PECy5 (Biolegend #301308), CD34‐APC (BD biosciences #555824), CD14‐APC‐Cy7 (Biolegend #325620), CD45RA‐PE (BD bioscience # 555489), CD11b‐PECy5 (Biolegend #301308), CD38‐PECy7 (BD biosciences #560677), in vivo engraftment levels were analyzed with anti‐human CD45‐APC (BD biosciences #555485), CD33‐PECy5 (BD biosciences #551377) and CD19‐PECy7 (BD biosciences #557835). Cell sorting was performed on BD FACS Aria II. Data were acquired on a BD LSRII or BD Celesta flow cytometer equipped with a High throughput sampler (HTS) device and analyzed using BD FACS Diva 4.0 and Flowjo X (Treestar Inc.) software.
8
Multiparameter Flow Cytometry of Hematopoietic Cells
9
Human Cell Differentiation and Engraftment Analysis
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hESC/OP9 and iPSC/OP9 cell cocultures were harvested and washed with FACS buffer (PBS with 2% FBS). Cells were analysed for HSC differentiation efficiency using an anti-human CD34-PE-Cy7 monoclonal antibody (BD). Mouse peripheral blood cells and femur BM cells were collected to analyse the human cell ratios. The cells were treated with erythrocyte lysate (E Bioscience), washed with FACS buffer and stained with the following monoclonal antibodies from BD Biosciences: anti-human CD3-FITC, anti-human CD8-PE-Cy7, anti-human CD31-PE, anti-human CD34-PE-Cy7, anti-human CD43-FITC, anti-human CD45-APC and anti-human CD71-PE. Staining of erythrocytes with anti-human CD235-APC monoclonal antibody (BD Biosciences) does not require lysis. Finally, the cells were resuspended in 200 μl of FACS buffer and analysed by flow cytometry on a weekly basis for 10 weeks after transplantation.
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