The mAbs anti-HIV-1 p24-FITC (clone KC57), anti-Vα24-FITC (clone C15), and anti-Vβ11-PE (clone C21) were from Beckman Coulter, anti-CD1d-PE (clone CD1d42), anti-CD3 AF700 (clone UCHT1), anti-CD4 BV605 (clone RTA-T4), anti-CD11c-allophycocyanin (clone B-ly6), anti-CD11c PE-Cy5 (clone B-ly6), anti-CD45 PerCP (clone 2D1), anti-CD56 AF700 (clone B159), anti-CCR5 APC-Cy7 (clone 2D7/CCR5), anti-DC-SIGN v450 (clone DCN46) and anti-HLA-DR APC (clone L243) were from BD Biosciences (San Jose, CA, USA), anti-CD4 BV711 (clone OKT4) and anti-CD8 BV570 (clone RPA-T8) were from BioLegend (San Diego CA), anti-CD14 PE-Texas red was from Invitrogen, and anti-CD19 PE-Texas red (clone SJ25-C1) was from Abcam (Cambridge, UK). Data were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo Version 9.7.5 software (TreeStar, Ashland, OR, USA). In some experiments, DCs were treated with 5 µg/ml Imiquimod (InvivoGen, Toulouse, France), 5 µg/ml Poly(I:C) (InvivoGen), 1 µg/ml ssRNA40LyoVec (Invivogen), 50 µM N-(n-Butyl)-deoxygalactojirimsin (NB-DGJ) (Toronto Research Chemicals, Toronto, Canada), 10 µM Chloroquine (InvivoGen), or with 5.6 µM TLR7 oligonucleotide antagonist IRS954: 5′-TGCTCCTGGAGGGGTTGT-3′ or the control oligo 5’-TCCTGCAGGTTAAGT-3 (Integrated DNA Technologies) (41 (link)).
Anti cd45 percp clone 2d1
Manufactured by BD
Sourced in United States
Anti-CD45 PerCP (clone 2D1) is a fluorochrome-conjugated monoclonal antibody that binds to the CD45 antigen, which is expressed on the surface of most human leukocytes. This product is suitable for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd146pe clone 128 018, by R&D Systems (2 mentions)
Anti vegf r2 kdr pe clone 89 106, by R&D Systems (2 mentions)
Lsr fortessa instrument, by BD (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Ssrna40 lyovec, by InvivoGen (1 mentions)
Chloroquine, by InvivoGen (1 mentions)
Anti hla dr apc clone l243, by BD (1 mentions)
Imiquimod, by InvivoGen (1 mentions)
Facsaria iiu cytometer, by BD (1 mentions)
Anti cd34 apc clone 581, by Thermo Fisher Scientific (1 mentions)
Navios flow cytometer, by Beckman Coulter (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
Anti hla dr fitc clone g46 6, by BD (1 mentions)
Anti cd8 pe cy7 clone rpa t8, by BD (1 mentions)
Anti cd56 pe clone my31, by BD (1 mentions)
Anti cd16 apc cy7 clone 3g8, by BD (1 mentions)
Anti cd11b pe clone icrf44, by BD (1 mentions)
Facsdiva software v7, by BD (1 mentions)
Facsdiva 7, by BD (1 mentions)
5 protocols using anti cd45 percp clone 2d1
1
Flow Cytometric Analysis of Dendritic Cells
2
Isolation and Characterization of MSCs
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To obtain MSC, reaming samples were collected from the intramedullary canal of the femoral head from 4 patients undergoing prosthetic hip replacement who attended the Department of Orthopedics and Traumatology of Hospital Universitario San Ignacio (Bogotá, Colombia). The sample donors participated voluntarily and accepted the informed consent previously reviewed and approved by the independent ethics committees (IECs) of the Hospital Universitario San Ignacio (Approval: 04/2020) in the ordinary session of 12 March 2020. MSCs were isolated and cultured according to protocols previously published by our group [44 (link),47 (link),48 (link)] and ISO 24651 international standard. MSC phenotype was confirmed by evaluating antigens as: CD34 (anti CD34-APC, clone 581, Thermo Scientific®, Waltham, MA, USA), CD45 (anti CD45-PerCP, clone 2D1, BD Biosciences®), CD73 (anti CD73-FITC, clone AD2, BD Pharmingen™, San Diego, CA, USA) and CD105 (anti CD105-PE, clone SN6, Thermo Scientific®) by flow cytometry using the FACSAria II-U cytometer (BD Biosciences®) and the data were analyzed using FlowJo 10.8.1 Software (Tree star, Ashland, OR, USA).
3
Comprehensive Immunophenotyping of Blood Samples
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Fresh BM samples were diluted 1:5 with PBS and analyzed with a hematocytometer to count white blood cells (WBCs), red blood cells (RBCs), and the percentage of neutrophils. Immunophenotyping was then performed using two calibrated mixtures of conjugated mAbs: the first evaluated the myeloid compartment, containing the mAbs anti-HLA-DR-FITC (clone G46-6, BD Biosciences, San Jose, CA, USA), anti-CD11b-PE (clone ICRF44, BD Bioscience), anti-CD45-PerCP (clone 2D1, BD Bioscience, San Jose, CA, USA), anti-CD14-PE-Cy7 (clone M5E2, BD Bioscience), anti-CD33-APC (clone WM53, BD Bioscience), and anti-CD16− APC-Cy7 (clone 3G8, BD Bioscience), and the second evaluated the lymphoid compartment, composed of the Abs anti-CD56-PE (clone MY31, BD Bioscience), anti-CD8-PE-Cy7 (clone RPA-T8, BD Bioscience), anti-CD3-APC (clone SK7, BD), anti-CD19-APC-Cy7 (clone SJ25C1, BD Bioscience), and anti-CD4-FITC (clone L200, BD Bioscience). Cells were stained for 30 min at 4 °C in the dark in PBS containing 2% FCS buffer, washed, and analyzed using a Navios Flow Cytometer (Beckman Coulter Inc. Brea, CA, USA).
4
Endothelial Progenitor Cell Characterization
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The characterization of ECFCs was performed using specific antibodies conjugated to fluorochromes (fluorescein isothiocyanate [FITC], phycoerythrin [PE] and peridin chlorophyll protein [PerCP]), which detect the following specific endothelial surface markers: anti‐CD31‐FITC, clone MBC 78.2 (Invitrogen); anti‐CD144‐PE, clone TEA 1/31 (Beckman Coulter); anti‐CD146PE, clone 128,018 (R&D Systems); anti‐VEGF R2/KDR‐PE, clone 89,106 (R&D Systems); anti‐CD34‐FITC, My10 clone (BD); anti‐CD45‐PerCP, clone 2D1 (BD); anti‐CD133‐APC, clone AC133 (Miltenyi Biotech). The tubes were incubated for 30 min at 4°C, protected from light, washed with PBS, centrifuged at 450 g for 5 min (RT) and resuspended in 300 μl of PBS for the acquisition of 10,000 events on a flow cytometer (FACS Calibur, Immunofluorometry Systems). Data analysis was performed using the BD FACS DIVA software (v.7.0; San Jose, CA, USA). The cells were considered ECFCs if they tested positive for CD31, CD144, CD146 and KDR markers, negative for CD45 and CD133, and exhibited decreased CD34 expression.
5
Phenotypic Characterization of Endothelial Colony-Forming Cells
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Characterization of ECFC was performed using speci c antibodies conjugated to uorochromes [ uorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP)] that detect speci c endothelial surface markers: anti-CD31-FITC, clone MBC 78.2 (Invitrogen, Camarillo, CA, USA), anti-CD144-PE, clone TEA 1/31 (Beckman Coulter, Marseille, France), anti-CD146PE, clone 128018 (R&D Systems, Minneapolis, MN, USA), anti-VEGF R2/KDR-PE, clone 89106 (R&D Systems, Minneapolis, MN, USA), anti-CD34-FITC, My10 clone (BD, San Jose, CA, USA), anti-CD45-PerCP, clone 2D1 (BD, San Jose, CA, USA), and anti-CD133-APC, clone AC133 (Miltenyi Biotech, Auburn, CA, USA). The cytometric tubes were incubated for 30 min at 4 °C, protected from light, washed with PBS, centrifuged at 450 g for 5 min (RT), and then re-suspended in 300 µL of PBS for the acquisition of 10,000 events on a cytometer ow (FACS Calibur, Immuno uorometry Systems, Mountain View, CA, USA). Data analysis was performed using BD FACS DIVA 7.0 software (San Jose, CA, USA). Cells were considered to be ECFC when they were positive for CD31, CD144, CD146, and KDR markers, negative for CD45 and CD133, and showed decreased CD34 expression.
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