The cell phenotype was assessed by fluorescence-activated cell sorting (FACS) on BD FACSAria flow cytometer (BD Pharmingen, BD Horizon USA). Staining with monoclonal antibodies (PerCP-Cy5.5 mouse antihuman CD105, APC mouse antihuman CD73, FITC mouse antihuman CD90, PE-Cy5 mouse antihuman HLA-DR, APC mouse antihuman CD34, and FITC mouse antihuman CD45) in accordance with manufacturer's instructions (BD Pharmingen, BD Horizon USA). The analysis was performed using BD FACS Diva 6.1 software (BD Pharmingen, BD Horizon USA).
Apc mouse anti human cd34
Manufactured by BD
Sourced in United States
The APC mouse anti-human CD34 is a laboratory reagent used for the identification and enumeration of CD34-positive cells in biological samples. CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells. This product is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Apc mouse anti human cd43, by BD (2 mentions)
Pe mouse anti human cd31, by BD (2 mentions)
Facsdiva 6, by BD (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Fitc mouse anti human cd90, by BD (1 mentions)
Percp cy5.5 mouse antihuman cd105, by BD (1 mentions)
Apc mouse antihuman cd73, by BD (1 mentions)
Pe cy5 mouse antihuman hla dr, by BD (1 mentions)
Fitc mouse anti human cd45, by BD (1 mentions)
Pe mouse anti human hla dr, by BD (1 mentions)
Influx cell sorter, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Pe mouse anti human cd29, by BD (1 mentions)
Fitc mouse anti human cd105, by BD (1 mentions)
Human regulatory t cell staining kit, by Thermo Fisher Scientific (1 mentions)
Anti cd4 fitc cd25 apc cocktail, by Thermo Fisher Scientific (1 mentions)
Human th1 th2 th17 phenotyping kit, by BD (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
11 protocols using apc mouse anti human cd34
1
Phenotyping Mesenchymal Stem Cells by FACS
2
Phenotypic Analysis of Immune Cell Subsets
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Collected MSCs were incubated for 30 min at room temperature with the following specific antibodies: PE mouse anti-human CD29, FITC rat anti-human CD44, FITC mouse anti-human CD105, FITC rat anti-human CD45, APC mouse anti-human CD34, and PE mouse anti-human HLA-DR (all from BD Pharmingen). As a control, the cells were stained with the appropriate isotype antibodies. At the end of co-culture, the CD4+ T cell apoptosis was analyzed by using an Annexin V-PE Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. To detect Treg cells, a Human Regulatory T Cell Staining Kit (eBioscience) containing an anti-CD4-FITC/CD25-APC cocktail and anti-Foxp3-PE was used according to the manufacturer’s instruction. In addition, we used a Human Th1/Th2/Th17 Phenotyping kit (BD Pharmingen) to analyze the T helper cell subsets. All samples were analyzed using a BD Biosciences Influx cell sorter.
3
Cytarabine and Danorubicin Potentiation
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Cytarabine (Ara‐C) and danorubicin were purchased from Sigma‐Aldrich (St. Louis, MI, USA). PTL and 3‐(1H‐1,2,3‐triazol‐4‐yl) pyridine (3‐TYP) were from Selleckchem (Houston, TX, USA). All compounds, except for in vivo studies, were reconstituted in dimethlysulfoxide, stored at 100‐mmol/l stock concentrations at −80°C, and used at the indicated doses suggested by the vendor. Flow cytometry antibodies, Alexa Fluor 647 Rabbit Anti‐Active caspase 3, PE‐Cy7 Mouse Anti‐Human CD38, APC‐H7 Mouse Anti‐Human CD45, and APC Mouse Anti‐Human CD34 were purchased from BD Pharmingen (San Jose, CA, USA). Immunoblotting antibodies, cleaved caspase 9, MCL1, BCL2, BAD, BAX, acetylated lysine and SIRT3 were purchased from Cell Signalling Technology (Danvers, MA, USA). Acetylated SOD2 and SOD2 antibodies were purchased from Abcam. ATPA (51) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
4
Cell Cycle Analysis and Neutrophil Differentiation
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Cell cycle profiles were analyzed by propidium iodide staining as described in detail elsewhere18 (link). The number of apoptotic cells in the population was estimated by the Annexin-V Alexa488/PI kit (Invitrogen) according to the provider’s recommendations. Maturation status of the differentiating neutrophilic cell population was analyzed by immuno-detection of cell surface markers. The following combination of antibodies were used: PE mouse anti-human CD16b, APC mouse anti-human CD34, APC-Cy7 mouse anti-human CD11b, PE-Cy7 mouse anti-human CD14 (all from BD Biosciences) and FITC-anti-Annexin V (Invitrogen). Neutrophilic differentiation stages were identified by gating on subpopulations according to the expression of surface markers (Supplementary Fig. S6 )57 (link): CD34+ cells (CD34+, CD11b−, CD16b−), promyelocytes (CD34−, CD11b−, CD16b−), myelocytes (CD34−, CD11b+, CD16b−) and metamyelocytes (CD36−, CD11b+, CD16b+). Monocytes were identified according to the expression of CD14+. All samples were measured with a FACS cytometer (BD Biosciences) and analyzed by the FlowJo software. Data were statistically analyzed by χ2 test for each replicate as well as pairwise comparison by Student’s t-test in GraphPad Prism. Full methodical details are given in the Supplementary information online.
5
Phenotypic Characterization of Mesenchymal Stem Cells
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The phenotype of MSC was confirmed prior to cell delivery by flow cytometry. Cells suspended in staining buffer containing 1% BSA and 0.05% sodium azide in D-PBS were incubated with Rat BD Fc Block (BD Biosciences, USA) for 5 min., and later with fluorochrome-conjugated antibodies: APC anti-human CD29, FITC anti-human CD44, BV421 anti-human CD90, APC-anti-human CD105, BV421 anti-human CD73 (BD Biosciences, USA), BV570 anti-human CD45 (Biolegend, USA), APC mouse anti-human CD34, APC mouse anti-human CD14 (BD Biosciences) for 40 min. Following washing with a cell staining buffer, cells were fixed with 1% neutral buffered formalin overnight. Samples resuspended in 1% BSA were assessed on the following day using a BD LSR II cell analyzer (Becton Dickinson, USA).
6
Immunophenotyping of Differentiated Cells
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The differentiated cells in T12.5 flask were dissociated into single cells using 0.05% trypsin supplemented with 0.1% EDTA. The cells were washed, resuspended in a FACS washing buffer (PBS with 5% BSA and 2.5 mM EDTA) and then stained with the desired antibodies. The antibodies used in our study were: PE Mouse Anti-Human CD31 (1:25, BD), FITC Mouse Anti-Human CD34 (1:25, BD), APC Mouse Anti-Human CD34 (1:25, BD), APC Mouse Anti-Human CD43 (1:25, BD), APC Mouse Anti-Human CD45 (1:25, BD), FITC-conjugated mouse IgG2a (1:25, BD), APC-conjugated mouse IgG1 (1:25, BD) and PE-conjugated mouse IgG1κ (1:25, BD) were used as isotype-matched negative controls. Flow cytometry was performed on Calibur flow cytometer (BD) or CytoFLEX V2-B4-R2 Flow Cytometer (BECKMAN COULTER), and the data was analyzed using FlowJo software, version 10.0.7.
7
Immunophenotyping of Progenitor Cells
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Cells were pretreated with Accutase (Invitrogen) for 3 minutes at 37°C and then washed with cold PBS. After 300×g centrifugation, 2×105 cells were resuspended in 100 ml of PBS and incubated with primary antibodies for 30 minutes at 4°C. Then, the cells were washed by using PBS with 2% FBS and 0.2 mM EDTA and used for flow cytometry analysis or sorting. The antibodies were as follows: Human APJ-APC-conjugated Antibody (R&D), PE Mouse Anti-Human CD31 (BD Biosciences), APC Mouse Anti-Human CD34 (BD Biosciences), APC Mouse Anti-Human CD43 (BD Biosciences), Mouse IgG3 APC-conjugated Antibody (R&D), PE Mouse IgG1, κ Isotype Control (BD Biosciences).
8
Isolation and Characterization of Human Thyroid-Derived Cells
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Ficoll-Paque PLUS was purchased from GE Healthcare Bio-Science (cat. no. 17-1440-03, Uppsala, Sweden). Dulbecco's modified Eagle's medium (DMEM; Cat. no. 11 965-092), fetal bovine serum (FBS; cat. no. 16 000-044), and penicillin-streptomycin mixture (cat. no. 15 140-122) were from Life Technology (Grand Island, NY, USA). Bovine TSH (bTSH; cat. no. 609385) came from EMD Millipore (Billerica, MA, USA). M22 activating anti-TSHR mAb was supplied by Kronus (Star, ID, Cat. no. M22-5c/00-690). Teprotumumab (Tepro, RV001) was a gift from River Vision Development (New York, NY, USA). APC mouse anti-human CD34 (Cat. no. 560940) was from BD Biosciences (San Jose, CA, USA). DEX (Cat. no. D4902) was from Sigma-Aldrich, LLC (St. Louis, MO, USA). SB203580 (Cat. no. 559389) was from Calbiochem/EMD Biosciences (Gibbstown, NJ, USA). 5, 6-dichloro-β-D-ribofuranosylbenzimidazole (DRB; Cat. no. 10 010 302) was from Cayman Chemical (Ann Arbor, MI, USA) while the pGL3 vector backbone was from GeneCopoeia (Rockville, MD, USA).
9
Immunophenotyping of Mesenchymal Stem Cells
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The immunophenotype characterization of the MSCs was conducted using a previously described protocol [1 (link)]. Conjugated monoclonal antibodies against PE-Cy™5 mouse anti-human CD90, PE-Cy™7 mouse anti-human CD73, APC mouse anti-human CD13, FITC mouse anti-human HLA-ABC, APC mouse anti-human CD34, FITC mouse anti-human CD31, PE mouse anti-human CD45, PE mouse anti-human CD14 (BD Biosciences, San Diego, CA, USA), eFluor™450 mouse anti-human CD105, PE mouse anti-human CD29 (eBioscience, San Diego, CA, USA), PE/Cyanine7 mouse anti-human HLA-DR and PE/Cyanine7 mouse anti-human CD10 (BioLegend, San Diego, CA, USA) were used.
10
Cell Cycle Analysis of iPS-HSPCs
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Cell cycle analysis of iPS‐HSPCs was carried out using the FITC BRDU Flow Kit (BD Pharmingen, San Jose, CA) following manufacturer's recommendations. 5′‐Bromo‐2′‐deoxyuridine (BrdU) substrate was added to iPS‐HSPC cultures 18 hours before collection for analyses; samples were prestained with mouse anti‐human CD34‐APC (BD; San Jose, CA, #555824) before fixation.
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