Integrin expression was assessed by flow cytometry using CD49a-phycoerythrin (CD49a-PE), CD49b-PE, CD49c-PE, CD49d-PE, CD49e-PE, CD49f-PE, CD29-allophycocyanin (APC) monoclonal antibodies (Pharmingen, San Diego, CA, USA) following manufacturer’s instructions. Nonspecific IgG of the corresponding class were used as isotype controls. ECM proteins fibronectin, from human plasma, collagen type IV, laminin, and vitronectin were from Sigma (St Louis, MO, USA). Matrigel was from BD Biosciences (San Jose, CA, USA). Native SPARC was purified from A375N human melanoma cells conditioned media.
Cd49a pe
Manufactured by BD
Sourced in United States
CD49a-PE is a fluorescently labeled antibody that binds to the CD49a cell surface antigen. It can be used for the identification and enumeration of cells expressing CD49a by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd49b pe, by BD (3 mentions)
Cd49e pe, by BD (3 mentions)
Cd49f pe, by BD (3 mentions)
Cd56 pe cy7, by BioLegend (2 mentions)
Cd3 bv510, by BioLegend (2 mentions)
Il 12, by Thermo Fisher Scientific (2 mentions)
Cxcr6 percp cy5, by BioLegend (2 mentions)
Il 15, by R&D Systems (2 mentions)
Isotype control, by BD (2 mentions)
Cd45 fitc, by BD (2 mentions)
Cd106 fitc, by BD (2 mentions)
Cd29 pe, by BD (2 mentions)
Cd44 apc, by BD (2 mentions)
Cd54 pe, by BD (2 mentions)
Cd324 pe, by BD (2 mentions)
Cd47 pe, by BD (2 mentions)
Cd62l apc, by BD (2 mentions)
Cd49c pe, by BD (2 mentions)
Cd49d fitc, by BD (2 mentions)
Cd51 61 pe, by BD (2 mentions)
6 protocols using cd49a pe
1
Integrin Expression Profiling by Flow Cytometry
2
Cytokine-Stimulated Immune Cell Profiling
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PBMCs and liver MNCs were stimulated for 12 h with IL‐12 10 ng/ml (PeproTech) or IL‐15 1 ng/ml (R&D Systems) to examine IFNγ and TNFα production respectively. An unstimulated control was included. BD GolgiStop™ (BD Biosciences) was added (4 µl/6 ml culture medium) for the last 4 h. Surface staining was performed for CD3‐BV510 (Biolegend®), CD56‐PE‐Cy7 (Biolegend®), CXCR6‐PerCP/Cy5.5 (Biolegend®), and CD49a‐PE (BD Biosciences). Cells were fixed and permeabilised (BD Cytofix/Cytoperm™ Plus Kit, BD Biosciences) prior to incubation with IFNγ (B27, APC, Biolegend®) or TNFα (MAb11, FITC, Biolegend®).
3
Surface Marker Profiling by Flow Cytometry
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Cell surface staining was performed
by the direct immunofluorescence assay with fluorescent-conjugated
antibodies: CD11c-APC (#17-0114-82), CD14-AF700 (#56-0149-42), and
CD54-FITC (#11-0541-82) from Thermo Fisher; CD49a-PE (#562115) and
CD44-FITC (#338803) from BD Biosciences; and corresponding isotype
control antibodies, for 30 min at 4 °C in PBS with 1% FBS, 1%
BSA, and 1% human serum (Sigma) to block Fc receptors. Cells were
analyzed in a FACSCanto II flow cytometer (Becton-Dickinson). The
results were analyzed using FlowJo.
by the direct immunofluorescence assay with fluorescent-conjugated
antibodies: CD11c-APC (#17-0114-82), CD14-AF700 (#56-0149-42), and
CD54-FITC (#11-0541-82) from Thermo Fisher; CD49a-PE (#562115) and
CD44-FITC (#338803) from BD Biosciences; and corresponding isotype
control antibodies, for 30 min at 4 °C in PBS with 1% FBS, 1%
BSA, and 1% human serum (Sigma) to block Fc receptors. Cells were
analyzed in a FACSCanto II flow cytometer (Becton-Dickinson). The
results were analyzed using FlowJo.
4
Characterization of PBMC and Liver MNC Activation
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Paired PBMCs and perfusate MNCs were resuspended in PBS/0.1% BSA to create a 2x cell solution. This was resuspended in Carboxyfluorecin succinimidyl ester (CFSE) staining solution (CellTrace™ CFSE Cell Proliferation Kit) (Life Technologies™, Paisely, UK) to make a final CFSE concentration of 5 μM and incubated for 10 min, 37°C. Staining was quenched with 5 volumes ice‐cold Roswell Park Memorial Institute Medium (RPMI) 1640 + Glutamax (Gibco®, Life Technologies™) supplemented with 10% fetal bovine serum (Hyclone®, Thermoscienticic, Northumberland, UK), penicillin, streptomycin and glutamine (Gibco®, Life Technologies™) (R‐10) and incubated for 5 min, 4°C. Cells were washed three times in R‐10 then recounted. PBMCs and liver MNCs were incubated in R‐10 supplemented with 5% HS (Sigma) in addition to Recombinant Human IL‐2 100 U/ml (PeproTech, London, UK), IL‐12 10 ng/ml (PeproTech), IL‐15 25 ng/ml (R&D Systems, Oxford, UK), IL‐18 100 ng/ml (Medical and Biological Laboratories, Japan), or a cocktail of all four for 6 days. Media and cytokines were changed every 2–3 days. A CFSE FMO was included. On day 0 and 6 PBMCs and liver MNCs underwent staining with Zombie Violet™ Fixable Viability Kit (Biolegend®), CD3‐BV510 (Biolegend®), CD56‐PECy7 (Biolegend®), NKG2C‐APC (Miltenyi Biotec), CXCR6‐PerCP/Cy5.5 (Biolegend®), and CD49a‐PE (BD Biosciences).
5
Multimarker Profiling of Cell Lineages
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The primary anti-human or anti-mouse antibodies (CD29-PE, CD49a-PE, CD49b-PE, CD49c-PE, CD49d-FITC, CD49e-PE, CD49f-PE, CD51/61-PE, CD44-APC, CD45-FITC, CD47-PE, CD324-PE, CD325-PE, CD326-PE, CD54-PE, CD106-FITC, CD62E-APC, CD62L-APC, CD15s, isotype controls) and secondary antibodies were all obtained from Becton Dickinson (BD) Pharmingen™. Human interleukin-1 beta (IL-1β) was purchased from Cell Signaling Technology, Inc. 17β-estradiol (E2) and progesterone (P4) were purchased from Shanghai Yuanye biological technology Co., Ltd Mifepristone (RU-486) was purchased from Shanghai New Hualian pharmaceutical Co., Ltd with purity > 98%. Metapristone was synthesized by our laboratory with purity > 98%. Calcein-AM was obtained from Dojindo Molecular Technologies, Inc.
6
Flow Cytometric Analysis of Cell Surface Markers
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The primary anti-human or anti-mouse antibodies (CD29-PE, CD49a-PE, CD49b-PE, CD49c-PE, CD49d-FITC, CD49e-PE, CD49f-PE, CD51/61-PE, CD44-APC, CD45-FITC, CD47-PE, CD324-PE, CD325-PE, CD326-PE, CD54-PE, CD106-FITC, CD62E-APC, CD62L-APC, CD15s, isotype controls) and secondary antibodies were all obtained from Becton Dickinson (BD) Pharmingen™. Human interleukin-1 beta (IL-1β) was purchased from Cell Signaling Technology, Inc. 17β-estradiol (E2) and progesterone (P4) were purchased from Shanghai Yuanye biological technology Co. Mifepristone (RU-486) was purchased from Shanghai New Hualian pharmaceutical Co., with purity > 98%. Metapristone was synthesized by our laboratory with purity > 98%. Calcein-AM was obtained from Dojindo Molecular Technologies, Inc.
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