The immunophenotype of cells cultured for two passages in human mesenchymal stem cells growth medium (MSCGM–Lonza, Verviers, Belgium) was analyzed by FACSCalibur™ (Becton-Dickinson, Franklin Lakes, NJ, USA) and CellQuest™ analysis software, after staining with the following conjugated antibodies: anti human CD45-PE, CD34-PE, CD31-PE, CD90-PE, CD73-PE, CD166-PE, CD44-PE (all from Becton Dickinson), and CD105-FITC (R&D System, Minneapolis, MN, USA). Cells were incubated with saturating concentrations of the appropriate antibodies for 30 min at 40 °C.
Anti human cd45 pe
Manufactured by BD
Sourced in United States
Anti-human CD45-PE is a fluorescent-labeled antibody that binds to the CD45 antigen expressed on the surface of human leukocytes. It is used for the identification and enumeration of CD45-positive cells in flow cytometric applications.
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Lab products found in correlation
Fitc anti human cd90, by BD (3 mentions)
Facscalibur, by BD (2 mentions)
7 aminoactinomycin 7 aad, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc, by BD (2 mentions)
Annexin 5 pe, by BD (2 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd45 fitc antibodies, by BD (2 mentions)
Anti human cd117 pe antibody, by BD (2 mentions)
Specific active caspase 3 antibody, by BD (2 mentions)
Anti cd3 apc, by BD (2 mentions)
Anti b220 pe, by BD (2 mentions)
Anti mac 1 apc, by BD (2 mentions)
Anti human lair1 clone 342219, by R&D Systems (2 mentions)
Anti gr 1 pe, by BD (2 mentions)
Pe conjugated anti annexin 5 and 7 aad, by BD (2 mentions)
Anti human cd3 fitc, by BioLegend (2 mentions)
Apc anti human cd33, by BioLegend (2 mentions)
Pe anti mouse cd8a, by BioLegend (2 mentions)
Pe anti mouse cd45, by BD (2 mentions)
Anti human cd8 pe, by BD (2 mentions)
22 protocols using anti human cd45 pe
1
Immunophenotyping of Cultured MSCs
2
Evaluation of Apoptosis and ROS in Primary Cells
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Apoptosis assays were performed as described previously (10 (link)). Briefly, after 24 to 48 hours of treatment, primary cells were stained for the surface antibodies CD34-allophycocyanin (APC), CD38-phycoerythrin with cyanin-7 (PECy7), CD123-phycoerythin (PE), and CD45-allophycocyanin-Hilite.7-BD (APC-H7; Becton Dickinson,) for 15 minutes. Cells were washed in cold PBS and resuspended in 200 μL of Annexin-V buffer (0.01mol/L HEPES/NaOH, 0.14 mol/L NaCl, 2.5 mmol/L CaCl2) containing Annexin-V-FITC or Annexin-V-PE (Becton Dickinson) and 7-aminoactinomycin (7-AAD, Life Technologies). Cells were incubated at room temperature for 15 minutes then analyzed on a BDLSRII flow cytometer using the high throughput attachment. To analyze human cell engraftment in the NOD/SCID xenotransplant model, bone marrow cells were blocked with the anti-Fc receptor antibody 2.4G2 and 25% human serum and later labeled with anti-human CD45-PE and anti-mouse CD45-FITC antibodies (Becton Dickinson). For reactive oxygen species (ROS) detection, the cells after treatment were incubated with 5 mmol/L CellROX Green Reagent (Life Technologies). The staining for CD117 was performed on MV4–11 cells after treatment with AR-42 or HSP90i using anti-human CD117-PE antibody (Becton Dickinson). Specific active caspase-3 antibody was used according to the manufacturer's protocols (BD Pharmingen).
3
Evaluation of Apoptosis and ROS in Primary Cells
4
Flow Cytometry Analysis of Mouse AML
5
Flow Cytometry Analysis of Mouse AML
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For flow cytometry analyses of mouse AML cells, peripheral blood or BM cells were stained with anti-Mac-1-APC (rat, M1/70, 1:200 dilution), anti-Gr-1-PE (rat, RB6-8C5, 1:200 dilution), anti-CD3-APC (hamster, 145-2C11, 1:200 dilution), anti-B220-PE (rat, RA3-6B2, 1:200 dilution), or anti-Kit-PE (rat, 2B8, 1:200 dilution) monoclonal antibodies (BD Pharmingen). For analysis of apoptosis, GFP or YFP positive AML cells were stained at indicated days with PE-conjugated anti-Annexin V and 7-AAD (BD Pharmingen) according to the manufacturer's instructions. For analysis of human hematopoietic engraftment in NSG mice, we followed our previously published protocol 53 (link), 54 (link) and used anti-human LAIR1 (clone 342219, R&D Systems) and anti-human CD45-PE (mouse, HI30, BD Pharmingen, 1:50 dilution) to quantify the total human AML engraftment.
6
Cytotoxicity and Flow Cytometry Analysis
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Analysis was done using a BD fluorescence-activated cell sorting (FACS) Canto II Flow Cytometer and BD LSRII Flow Cytometer (BD Biosciences, San Jose, CA). Data was analyzed using the BD FACSDiva software. Antibodies used included anti-CD69 APC-Cy7, anti-CD5 PerCP/Cy5.5, antihuman CD45 PE (BD Biosciences) and anti-myc tag fluorescein isothiocyanate (FITC,Abcam, Cambridge, MA). For the cytotoxicity studies, target cells were stained with the membrane dye PKH26 and cell death was assessed using 7-AAD (described below). Flow sorting for GFP was performed using a BD FACS Aria II Cell Sorter (BD Biosciences).
7
Multiparameter Flow Cytometry Analysis
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Primary antibodies including anti-mouse CD8a-PE (BioLegend, 100707, 53–6.7, 1:100), anti-mouse CD45-PE (BD Pharmingen,561087, 30-F11, 1:100), anti-mouse CD45.1-FITC (BioLegend, 110705, A20, 1:100), anti-mouse CD45.2-PE (BioLegend, 109807,104, 1:100), anti-human CD45-PE (BD Pharmingen, 555483, HI30, 1:100), anti-human CD33-APC (Biolegend,366605, P67.6, 1:100), anti-human CD3-FITC (BioLegend, 300305, HIT3a, 1:100), anti-human CD8-PE (BD Pharmingen, 555367, RPA-T8, 1:100) antibodies were used. Cells were run on Calibur for analysis or FACSAria for sorting.
8
Murine Lymphocyte Immunophenotyping
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Peripheral blood of mice was treated with 1 ml red blood cell lysis buffer (BioLegend Cat#420301) at room temperature for 10 min. Then the cells were centrifuged at 300 g, washed twice with PBS, resuspended in PBS, and stained with antibodies including anti-human CD45-PE (BD Biosciences Cat#555483), CD3-PerCP-Cy5.5 (BD Biosciences Cat#560835) and CD20-FITC (BD Biosciences Cat#555622) at 1:100 dilution for 30 min at 4 °C. After washing with PBS, the percentage of CD3+ or CD20+ cells among the CD45+ cells was quantified using a flow cytometer.
9
Submandibular Gland Cell Isolation
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Human submandibular gland samples were placed in a C tube (Miltenyi Biotec, North Rhine-Westphalia, German) containing digestion buffer (Multi Tissue Dissociation Kit 1, Miltenyi Biotec), and tissues were dissociated by a gentleMACS™ octo dissociator with heaters (Miltenyi Biotec). Submandibular gland cells in cell suspension were separated using Percoll solution (GE Healthcare, Chicago, IL). Subsequently, cells were stained with anti-human CD45 PE (BD biosciences, Franklin Lakes, NJ) antibodies. Non-immune cells (CD45-) were purified from stained cells using a BD Aria III (BD).
10
Quantifying Human AML Engraftment in NSG-S Mice
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Human AML engraftment was assessed by flow cytometry and defined as the percentage of human CD45+/CD33+ cells in total live mononuclear cells. Fresh bone marrow cells from NSG-S mice engrafted with K562-Luc and primary AML cells (6373) were collected at day 10 and 40 post-engraftment, respectively, once the blast cells were detected in peripheral blood smear. Samples were stained with antibodies for cell surface markers: anti-human CD45-PE and anti-human CD33-FITC (BD Biosciences, San Jose, CA, USA). Cells were incubated with monoclonal antibodies for 15 min at room temperature, washed once in PBS containing 0.1% human serum albumin (HSA), and analyzed by flow cytometry. Data acquisition was performed using a FACS Aria III (BD Biosciences) equipped with an argon and red diode laser, and analysis was performed using Cell Quest software (BD Biosciences).
As the K562-Luc cells express dim CD34, immunohistochemistry was performed for the formalin-fixed decalcified femurs from primary AML cells (6373) transplanted mice, paraffin-embedded and sectioned at 5-µm sections. Slides were stained using human anti-CD34 primary antibody (R&D System, Minneapolis, MN, USA), then with HRP-conjugated secondary antibody (Cell Signaling Technology Inc. Danvers, MA, USA). HRP activity was detected by diaminobenzidine tetrahydrochloride (DAB), and the slides were counterstained by methyl green.
As the K562-Luc cells express dim CD34, immunohistochemistry was performed for the formalin-fixed decalcified femurs from primary AML cells (6373) transplanted mice, paraffin-embedded and sectioned at 5-µm sections. Slides were stained using human anti-CD34 primary antibody (R&D System, Minneapolis, MN, USA), then with HRP-conjugated secondary antibody (Cell Signaling Technology Inc. Danvers, MA, USA). HRP activity was detected by diaminobenzidine tetrahydrochloride (DAB), and the slides were counterstained by methyl green.
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