TNCs were stained for hematopoietic lineage markers using the following fluorescein isothiocyanate (FITC)-conjugated antibodies (Abs) against human proteins:CD2 (clone RPA-2.10); CD3 (clone UCHT1); CD14 (clone M5E2); CD16 (clone 3G8); CD19 (clone HIB19); CD24 (clone ML5); CD56 (clone NCAM16.2); CD66b (clone G10F5); and CD235a (clone GA-R2) (all from BD Biosciences). The cells were simultaneously stained for the pan-leukocytic marker, CD45, with phycoerythrin (PE)-conjugated Abs (clone HI30; BD Biosciences) and one of the following Abs:allophycocyanin (APC)-conjugated CD34 (clone 581; BD Biosciences) or APC-conjugated CD133 epitope 1 (CD133/1; Miltenyi Biotec). Additionally, the following isotype controls were used:FITC-conjugated mouse IgG1 (clone MOPC-21), mouse IgG2a (clone G155-178), and mouse IgG2b (clone 27–35); PE-conjugated mouse IgG1 (clone MOPC-21); and APC-conjugated mouse IgG1 (clone MOPC-21) (all from BD Biosciences). In addition, the APC-conjugated isotype control mouse IgG1 (clone IS5-21F5; Miltenyi Biotec) was used.
Apc conjugated mouse igg1 clone mopc 21
Manufactured by BD
Sourced in United Kingdom
APC-conjugated mouse IgG1 (clone MOPC-21) is a monoclonal antibody conjugated with Allophycocyanin (APC). It is a laboratory reagent used for flow cytometry applications.
Automatically generated - may contain errors
3 protocols using apc conjugated mouse igg1 clone mopc 21
1
Hematopoietic Lineage Marker Profiling
2
Comprehensive Immune Phenotyping by Flow Cytometry
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The following mouse monoclonal anti-human antibodies were used for flow cytometry experiments: fluorescein isothiocyanate (FITC)-conjugated CD1b (clone M-T101), CD95 (clone DX2), and HLA-DPDQDR (clone Tu39); PE-conjugated CD119 (clone GIR-208), CD124 (clone hIL4R-M57), and CD132 (clone AG184); APC-conjugated CD1a (clone HI149), CD40 (clone 5C3), CD49d (clone 9F10), and HLA-ABC (clone G46-2.6; BD Biosciences, San Jose, CA, USA); FITC-conjugated CD54 (clone RR1/1); PE-conjugated CD147 (clone 8D12) (eBioscience, San Diego, CA, USA). The following isotype-matched control antibodies were also included in all experiments: FITC-conjugated mouse IgG1 (clone MOPC-21), IgG2a (clone G155-178); PE-conjugated mouse IgG1 (clone MOPC-21); and APC-conjugated mouse IgG1 (clone MOPC-21) (BD Biosciences).
3
Characterization of Cell Surface Markers
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Cells cryopreserved at Passage 3 from each donor were plated in proliferation
medium in 6 well tissue culture plates at a density of 5000 cells/cm2and cultured until they reached 80%–90% confluence. Cells were harvested from
the wells, as previously detailed, centrifugated at 1000×g and
fixed using 4% formaldehyde. Cells were stained with FITC-conjugated anti-CD56
(Clone AF-7H3, Milteny Biotec), BV421-conjugated anti-CD34 (Clone 581, BD
Biosciences) and APC-conjugated anti-CD90 (Clone 5E10, BD Biosciences) for 20
min in the dark at 4°C. Controls for non-specific staining were included
consisting of isotype controls for each antibody (FITC-conjugated IS5-21F5 mouse
IgG1 (Milteny Biotec, United Kingdom), BV421-conjugated anti-KLH (BD
Biosciences) and APC-conjugated mouse IgG1 clone MOPC-21 (BD Biosciences). Cells
were washed 2 times before the analysis. Each acquisition file included 10,000
events. A forward scatter (FSC) threshold was set to avoid debris from list mode
data and for each sample. Flow cytometry analysis was conducted using BD
LSRFortessa™ Cell Analyser (BD Biosciences) and BD FACSDiva™ Software for
acquisition. Results were analysed using FlowJo™ version 10 software (BD
Biosciences).
medium in 6 well tissue culture plates at a density of 5000 cells/cm2and cultured until they reached 80%–90% confluence. Cells were harvested from
the wells, as previously detailed, centrifugated at 1000×g and
fixed using 4% formaldehyde. Cells were stained with FITC-conjugated anti-CD56
(Clone AF-7H3, Milteny Biotec), BV421-conjugated anti-CD34 (Clone 581, BD
Biosciences) and APC-conjugated anti-CD90 (Clone 5E10, BD Biosciences) for 20
min in the dark at 4°C. Controls for non-specific staining were included
consisting of isotype controls for each antibody (FITC-conjugated IS5-21F5 mouse
IgG1 (Milteny Biotec, United Kingdom), BV421-conjugated anti-KLH (BD
Biosciences) and APC-conjugated mouse IgG1 clone MOPC-21 (BD Biosciences). Cells
were washed 2 times before the analysis. Each acquisition file included 10,000
events. A forward scatter (FSC) threshold was set to avoid debris from list mode
data and for each sample. Flow cytometry analysis was conducted using BD
LSRFortessa™ Cell Analyser (BD Biosciences) and BD FACSDiva™ Software for
acquisition. Results were analysed using FlowJo™ version 10 software (BD
Biosciences).
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