For analysis of cell-surface expression of hMRC1, cells were washed twice with ice-cold 20 mM EDTA-PBS, followed by two washes in ice-cold 1% BSA-PBS. Cells were stained in 1% BSA-PBS for 30 min at room temperature in the dark with allophycocyanin (APC)-conjugated anti-human CD206 antibody (α-hMRC1; BioLegend, San Diego, CA, USA) or APC-conjugated Mouse IgG1 (BD Biosciences Immunocytometry Systems, Mountain View, CA, USA) as isotype control. Cells were then washed twice with ice-cold 1% BSA-PBS and fixed with 1% formaldehyde in PBS. Finally, cells were analyzed on a FACSCalibur (BD Biosciences Immunocytometry Systems, Mountain View, CA, USA). Data analysis was performed using FlowJo (Tree Star, San Carlos, CA, USA).
Apc conjugated mouse igg1
Manufactured by BD
Sourced in United States
APC-conjugated mouse IgG1 is a fluorescent-labeled antibody used as a control in flow cytometry experiments. It binds non-specifically to cells and is used to establish background fluorescence levels.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (3 mentions)
Apc conjugated anti human cd206 antibody α hmrc1, by BioLegend (2 mentions)
Pe conjugated mouse igg1, by BD (2 mentions)
Permeabilizing solution, by BD (1 mentions)
Lysing solution, by BD (1 mentions)
Cd45ra, by BioLegend (1 mentions)
Anti human cd3 apc h7, by BD (1 mentions)
Anti human cd107a apc, by BD (1 mentions)
Anti human ifn γ pe cy7, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Pe conjugated mouse igg1, by R&D Systems (1 mentions)
Mouse igg2b, by Thermo Fisher Scientific (1 mentions)
Cd133, by Miltenyi Biotec (1 mentions)
7 aad, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Bv421 conjugated mouse igg1, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Apc mouse anti human cd34, by BD (1 mentions)
Apc mouse anti human cd43, by BD (1 mentions)
6 protocols using apc conjugated mouse igg1
1
Quantifying Cell-Surface hMRC1 Expression
2
Quantifying Cell-Surface hMRC1 Expression
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For analysis of cell-surface expression of hMRC1, cells were washed twice with ice-cold 20 mM EDTA-PBS, followed by two washes in ice-cold 1% BSA-PBS. Cells were stained in 1% BSA-PBS for 30 min at room temperature in the dark with allophycocyanin (APC)-conjugated anti-human CD206 antibody (α-hMRC1; BioLegend, San Diego, CA, USA) or APC-conjugated Mouse IgG1 (BD Biosciences Immunocytometry Systems, Mountain View, CA, USA) as isotype control. Cells were then washed twice with ice-cold 1% BSA-PBS and fixed with 1% formaldehyde in PBS. Finally, cells were analyzed on a FACSCalibur (BD Biosciences Immunocytometry Systems, Mountain View, CA, USA). Data analysis was performed using FlowJo (Tree Star, San Carlos, CA, USA).
3
Phenotypic Analysis of Activated Vγ9Vδ2 T Cells
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Vγ9Vδ2 T cells were stimulated with 5 μg/mL plate-bound anti-CD3 antibodies and 1 μg/mL soluble anti-CD28 antibodies (free mAbs) for 6 h in the presence of 5 μg/mL Brefeldin A. Then, the cells were stained with anti-human CD3-APC-H7 (BD Biosciences, clone: SK7), anti-human TCR Vδ2-PerCP (BioLegend, clone: B6), and anti-human CD107a-APC (BD Biosciences, clone: H4A3) antibodies. After staining for surface markers, the cells were fixed and permeabilized using Lysing Solution (BD Biosciences) and Permeabilizing Solution (BD Biosciences), respectively. Subsequently, the cells were stained with anti-human IFN-γ-PE-Cy7 (BD Biosciences, clone: B27), anti-human TNF-α-PE (BD Biosciences, clone: MAb11), and their corresponding isotype controls (APC-conjugated mouse IgG1, κ isotype control (clone: MOPC-21); PE-Cy™7-conjugated mouse IgG1, κ isotype control RUO (clone: MOPC-21); and PE-conjugated mouse IgG1, κ isotype control (clone: MOPC-21), all from BD Biosciences). Then, the cells were analyzed with a BD FACS Verse, and the data were analyzed with FlowJo. In addition, cell cycle, cell proliferation (Ki-67, BioLegend, clone: Ki-67), cell differentiation (CD45RA, BioLegend, clone: HI100), CD27 (BioLegend, clone: O323), mitochondrial, and costimulatory molecule (CD80, BioLegend, clone: 2D10; CD86, BioLegend, clone: BU63) analyses were performed following standard protocols.
4
Multiparameter Flow Cytometry Analysis
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Cells (5 × 105) were labeled with the following human antibodies: phycoerythrin (PE)-conjugated CD166 (ALCAM; BD Bioscience, San Jose, CA), CD324 (EpCAM; eBioscience, San Diego, CA), CD133 (Miltenyi Biotec, Bergisch Gladbach, German), CD44 (eBioscience), fluorescein isothiocyanate (FITC)-conjugated CD44 (eBioscience), biotin-conjugated CD24 (eBioscience), CD133 (Miltenyi Biotec), allophycocyanin (APC)-conjugated CD13 (eBioscience), CD133 (Miltenyi Biotec), and CD90 (eBioscience). The following isotype-matched mouse or rat immunoglobulins were used as controls: APC-conjugated mouse IgG1 (BD biosciences), mouse IgG2b (eBioscience), PE-conjugated mouse IgG1 (R&D Systems Inc., Minneapolis, MN), FITC-conjugated rat IgG2b (R&D Systems Inc.), biotin-conjugated mouse IgG1 (R&D Systems Inc.). Cell samples were analyzed by flow cytometry using a FACSCalibur (BD biosciences) and CellQuest software (Version 6.0, BD biosciences). 7-AAD (BD biosciences) was used to identify dead cells.
5
Flow Cytometry Immunophenotyping of PBMCs
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PBMCs and CD4+ T cells were collected and resuspended with 3% FBS on ice for 10 min. Hundred microliter cell suspension (1 × 106) was prepared for one test. Two microliter BV421 conjugated mouse anti-human CD25 (BD biosciences, 562443, USA), BB515 conjugated mouse anti-human CD4 (BD biosciences, 564419, USA) and APC conjugated mouse anti-human CD69 (BD biosciences, 560967, USA) Abs or PE conjugated mouse anti-human CXCR3 Ab (BD biosciences, 560928, USA) were added into the corresponding samples, followed by incubation on ice for 15 min. Background staining was assessed by isotype-matched control Abs, including BV421 conjugated mouse IgG1 (BD biosciences, 562438, USA), BB515 conjugated mouse IgG1 (BD biosciences, 564416, USA), APC conjugated mouse IgG1 (BD biosciences, 555751, USA), and PE conjugated mouse IgG1 (BD biosciences, 555749, USA). Cells were washed with 1 × PBS for three times. Three hundred microliter cell suspension was filtrated through a 200-mesh membrane and performed on BD LSRFortessa™ Flow Cytometer. Data were analyzed using BD FACSDiva software (BD Biosciences).
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.
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