Bone marrow cells were harvested from femurs and tibia of mice in alpha-MEM media (Corning Cellgro). For mice, freshly collected bone marrow cells were washed with phosphate buffered saline (PBS) and stained with lineage-specific, Scal-1, c-Kit and BrdU antibodies (BD Biosciences) according to manufacturer’s instructions. Analysis was performed using BD FACS diva on LSR II. Human Lin−CD184+CD45− mesenchymal stem/progenitor cells in the peripheral blood mono-nucleated cell population were identified using human hematopoietic lineage FITC cocktail, anti-human CD45 APC and anti-human CD184-PE (eBioscience, #22-7778-72, #17-9459-42, #12-9999-42).
Anti human cd45 apc
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-human CD45 APC is a fluorescently labeled antibody that specifically binds to the CD45 cell surface antigen expressed on human leukocytes. It is designed for use in flow cytometry applications to identify and quantify different subsets of white blood cells.
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Lab products found in correlation
Mem alpha media, by Corning (1 mentions)
Collagenase, by Merck Group (1 mentions)
Anti human integrin αvβ3 pe, by Abcam (1 mentions)
Canto 2, by BD (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
Anti mouse cd45 fitc, by Thermo Fisher Scientific (1 mentions)
Fitc anti human cd34, by BioLegend (1 mentions)
Annexin 5 fitc apoptosis detection kit, by 4A Biotech (1 mentions)
Facscalibur, by BD (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Facs canto 2 flow cytometer, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
5 protocols using anti human cd45 apc
1
Isolation and Identification of Murine and Human Mesenchymal Stem/Progenitor Cells
2
Integrin αvβ3 Expression Analysis
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Cells were collected in PBS and stained with anti-human integrin αvβ3 PE (1:100, Abcam, ab7166, Cambridge, U.K.) at 4°C for 30 min. To examine the percentage of integrin αvβ3 in patients’ samples, 1 mg/ml collagenase (Sigma–Aldrich, San Francisco, U.S.A.), 2 units/ml hyaluronidase (Sigma–Aldrich, San Francisco, U.S.A.), and 0.1 mg/ml DNase (Sigma–Aldrich, San Francisco, U.S.A.) were used to digest the tissues into single cells, the anti-human CD45 APC (1:100, eBioscience, CA, U.S.A.) and anti-human integrin αvβ3 PE (1:100, Abcam, Cambridge, U.K.) were stained at 4°C for 30 min. After washing, the data were collected by BD Canto II (BD Biosciences, NY, U.S.A.). 7-AAD was used to exclude the dead cells. IgG (1:1000, Abcam, Cambridge, U.K.) was used as the negative control. For integrin αvβ3+ cell sorting, BD FACSARIA III was used to sort PE positive cells.
3
Comprehensive Cell Analysis Protocol
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FACS analysis was performed routinely by using a CaliburTM flow cytometer (BD Immunocytometry Systems). Anti-mouse CD45-FITC (#104, eBioscience), anti-human CD45-APC (HI30, eBioscience), anti-human CD34-FITC (#581, Biolegend). Cell-cycle analysis was performed using DNA binding dye propidiumiodide (PI). Hematopoietic cells were fixed in 50 % ethanol and resuspended to 0.2 mL of 10 mg/mL RNAaseA and 50 µg/mL PI. Cell-cycle kinetics was performed with routine protocols using the FACS Calibur flow cytometer (Becton–Dickinson, CA). Apoptosis was analyzed by using an Annexin V-FITC Apoptosis Detection Kit (4A Biotech, Beijing, China).
4
Hematopoietic Differentiation of hESCs
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Hematopoietic differentiation was induced as described by Ji et al.49 (link). Briefly, the hESCs lines were transferred onto OP9 feeders for 15 days. To evaluate hematopoietic differentiation and eGFP expression at the different days, cells were dissociated with collagenase IV and Tryple (Gibco), resuspended in FACS buffer, filtered through a 70 μm cell strainer (BD Biosciences, Bedford, MA) and stained with anti-mouse CD29-FITC (AbD Serotec, Raleigh, GBK), anti-human CD34-PE-Cy7 and anti-human CD45-APC (all from eBiosciences, San Diego, CA) and analyzed in a FACS Canto II flow cytometer.
5
Xenograft model for T-ALL and B-ALL
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Human leukemic bone marrow cells from NOTCH1 mutant T-ALL patient 389E and BCR-ABL1 positive B-ALL patient XC51 were injected into the tail vein of 6-to-12-week old NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. After successful engraftment, splenocytes were harvested and reinjected at a concentration of 1-2 x10 6 cells into 30 NSG-mice to create secondary transplants. Blood samples were taken once a week and measured on the Vet ABC TM Hematology Analyzer (SCIL). Leukemic engraftment was determined with flow cytometry (FACS Canto II, BD) using anti-human CD45 (APC, eBioscience) as marker for the leukemic cells.
Treatment started when the CD45-percentage in the peripheral blood was respectively <0.5% and >5%. Mice were divided into groups of 4-5 mice, each treated animal matching a control mouse in both gender and CD45%. At the end of treatment, mice were sacrificed and spleen and bone marrow harvested for single cell isolation and histopathologic examination. All experiments were conducted on protocols approved by the ethical committee of the University of Leuven.
Treatment started when the CD45-percentage in the peripheral blood was respectively <0.5% and >5%. Mice were divided into groups of 4-5 mice, each treated animal matching a control mouse in both gender and CD45%. At the end of treatment, mice were sacrificed and spleen and bone marrow harvested for single cell isolation and histopathologic examination. All experiments were conducted on protocols approved by the ethical committee of the University of Leuven.
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