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.
Cd49d pe
Manufactured by BD
Sourced in United States, Germany
CD49d-PE is a fluorescently-labeled monoclonal antibody that binds to the CD49d (α4 integrin) cell surface antigen. CD49d-PE can be used for the identification and enumeration of CD49d-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd73 pe, by BD (4 mentions)
Cd90 apc, by BD (2 mentions)
Cd34 pe, by BD (2 mentions)
Collagen type 4, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
Vitronectin, by Merck Group (1 mentions)
Cd49a pe, by BD (1 mentions)
Cd49b pe, by BD (1 mentions)
Cd49e pe, by BD (1 mentions)
Cd49f pe, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Cd146 pe, by BD (1 mentions)
Cd45 fitc, by Miltenyi Biotec (1 mentions)
Cd166 pe, by BD (1 mentions)
Cd105 pe, by R&D Systems (1 mentions)
Cd106 pe cy5, by BD (1 mentions)
Hla abc pe, by Immunotools (1 mentions)
Cd34 apc, by Immunotools (1 mentions)
8 protocols using cd49d pe
1
Integrin Expression Profiling by Flow Cytometry
2
Multiparametric Characterization of Mesenchymal Stem Cells
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ASCs were further expanded in vitro and were analyzed at passages 4 to 5 by flow cytometry (FACSAria; BD Biosciences, Erembodegem, Belgium). Monoclonal antibodies against CD9‐PE, CD10‐PECy7, CD13‐PE, CD14‐PECy, CD19‐PECy7, CD29‐APC, CD49d‐PE, CD73‐PE, CD90‐APC, CD106‐PE‐Cy5, CD146‐PE, and CD166‐PE (BD Biosciences); CD45‐FITC (Miltenyi Biotech, Bergisch Gladbach, Germany); CD31‐FITC, CD34‐APC, CD44‐FITC, HLA‐ABC‐PE, and HLA‐DR‐PE (Immunotools GmbH, Friesoythe, Germany); and CD105‐PE (R&D Systems Inc., Minneapolis, Minnesota) were used. Analysis was performed on 10,000 cells per sample. The positive expression was defined as the level of fluorescence greater than 99% of the corresponding unstained cell sample.
3
Characterization of Lymphocyte Populations
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Blood samples were freshly analyzed by lyse-and-wash whole blood staining procedure. Surface staining was performed on ice for 20 min, and the cells were then analyzed on a two laser, six color FACSCanto flow cytometer. Multiplexed dilutions of monoclonal antibodies (mAbs) were used to characterize lymphocyte populations. The following antibodies from BD Biosciences were used: CD69-FITC, HLA-DR-FITC, CD49d-PE, CD11a-PE-Cy7, CD4APC-H7, CD8 APC-H7, CCR6-PerCP-Cy5.5, CD183 PE-Cy 7, CD44- PerCP-Cy 5.5.
4
Multiparametric Characterization of HUCB and Bone Marrow Cells
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Antibodies against the human antigens CD13-PE, CD31-PE, CD34-PE, CD44-fluorescein isothiocyanate (FITC), CD49d-PE, CD57-FITC, CD73-PE, CD90 (Thy-1)-pure, and CD105 (Endoglin)-pure were purchased from BD Biosciences (San Jose, CA), CD45-FITC was from Immunotech (Marseille, France), and CD133/1-PE, and CD271(p75NTR)-FITC, -PE, -APC were obtained from Miltenyi Biotec. For each antibody expression assessment, a total of 1 × 105 MNCs from HUCB and human bone marrow were resuspended in 100 μl PBS containing 0.5% BSA and 200 mM EDTA, incubated with rabbit serum for blocking nonspecific binding. The cells were then incubated with primary antibodies for 30 min on ice. Binding of unconjugated anti-CD90 and CD105 was detected by secondary staining with PE-conjugated anti-mouse IgG antibody (BD Biosciences). FITC- and PE-conjugated mouse IgGs were used as the control isotype at the same concentration as specific primary antibodies. The fluorescence intensity of the cells was evaluated by flow cytometry (FACSCalibur, BD Biosciences), and data was analyzed with CellQuest software (BD Biosciences).
5
Immunophenotyping of MSC Subtypes
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MSC marker expression in the PSP-MSCs and RA-Pα-MSCs was investigated by flow cytometry using the FACS Aria III, following incubation for 30 min at 4°C with the following monoclonal antibodies: CD34-FITC, CD45-APC, CD49d-PE, CD73-PE, CD90-APC, CD106-APC (BD Pharmingen), CD105-biotin, CD140α-biotin, and CD140β-biotin (Biolegend), CD271 (BD pharmingen), anti-VIMENTIN and anti-STRO1 (Abcam). Streptavidin APC (Bioscience) was used as the secondary antibody for the biotin-labeled primary antibodies.
6
Isolation and Characterization of Adipose Stem Cells
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Adipose stem cells were isolated from adipose tissue collected from hospital with the consent of patient (n = 3) going through liposuction. Isolation of adipose stem cells was performed following a previous protocol with slight modifications [43 ]. Briefly, the collected sample was washed thrice with 1× PBS. Washed adipose was treated with Collagenase 1A and incubated at 37°C for 45 minutes. Digested adipose was first filtered through a 100 µm mesh, treated with active media and then centrifuged for 10 minutes at 1200×g. SVF obtained in the pallet form was resuspended in 1 ml low-glucose Dulbecco’s modified eagle’s medium (LG-DMEM) and shifted to a 75cm2 flasks. Cells from passage 2 were further seeded for experimental purposes. Moreover, passage 2 cell were also subjected to Immunophenotyping through flow cytometery for CD 90-PE, CD73-PE, CD49D-PE, CD45-FITC, CD34-PE, CD106-FITC (BD Biosciences, USA) and CD105 (Santa Cruz Biotechnology, USA) according to minimal criteria of ISCT mentioned before for defining MSCs [11 (link)]. FACS were performed according to the already published protocol [44 (link)]
7
Flow Cytometry of Memory T Cell Subsets
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Samples of fresh ACD-anticoagulated whole blood were stained with monoclonal antibodies, lysed and fixed, as above. Monoclonal antibodies used to study subsets of CD4 memory T cells were: CD3-PerCP-Cy5.5, CXCR3-Alexa Fluor 488 and -PE-CF594, integrin ß7-APC, CD4-Alexa Fluor 700, CD45RO-FITC, CD45RA-PE-CF594; CD49d-PE, CD25-PE-Cy5, CD8-BV786 (BD Biosciences), CD45RO-ECD (Beckman Coulter); CD49d-BV510, integrin ß7-biotin, CCR6-BV421, CD127-PE-Cy7 (BioLegend); CD161-PE, -APC or PE-Vio-770 (Miltenyi Biotec, Bergisch Gladbach, Germany); and CD62 L-APC-Cy7 (eBiosciences). A total of 400,000 events were collected, and a manual forward and side scatter gate was drawn to include lymphocytes, then gated sequentially on CD31, on CD41, and then on CD45RO1 cells using FlowJo, then exported as an FCS file using FlowJo.
8
Phenotypic Characterization of CD34+ Cells
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Totally, 5 × 10 5 CD34+ cells were collected on days 0, 3, 7, 10, and 14. After washing twice with PBS, the cells were labeled using the following monoclonal antibodies: mouse antihuman CD34-FITC, CD42b-PE, CD41a-PE, CD61-FITC, CD49d-PE, CD49e-FITC, CD11a-FITC, CD54-PE, and mouse IgG 1 -FITC, IgG 2 -PE (BD, Pharmingen, USA). Cells in PBS at a volume of 100 with 10 μL of monoclonal antibody were incubated at 25°C for 30 min. After washing with PBS, the cells were resuspended in 100 μL of PBS and detected by FCM (Guava easyCyte 8HT, EMD Millipore, Billerica, MA, USA), and data were analyzed by Guava InCyte software (Version 2.8, EMD Millipore, Billerica, MA, USA).
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