fMSCs were dissociated with 0.05% trypsin-EDTA at 37 °C for 5 min. The ovary tissues were dissected, washed in PBS, and then enzymatically digested to single-cell suspensions (0.25% trypsin-EDTA for 15 min). Single-cell suspensions were passed through a 70-μm nylon filter (BD, USA). After washing with PBS, the collected cells were suspended in buffer (0.1% BSA in PBS). To analyze cell surface markers on fMSCs, the dissociated cells were stained with the following PE-conjugated antibodies: anti-CD105, anti-CD29, anti-CD73, anti-CD90, anti-CD34, and anti-CD45, all purchased from Becton Dickinson and Company (USA). The hGCs and cell suspensions from ovarian tissues were stained with anti-KI67 (BD, USA), anti-ROS (Abcam, USA), or its corresponding isotype control at 4 °C for 30 min. The stained cells were analyzed with a flow cytometer (Beckman, USA) using the manufacturer’s directions.
Anti cd29
Manufactured by BD
Sourced in United States
Anti-CD29 is a monoclonal antibody that recognizes the CD29 antigen, also known as the beta-1 integrin subunit. CD29 is expressed on various cell types and plays a role in cell adhesion and migration. This antibody can be used for the identification and isolation of cells expressing CD29 in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd90, by BD (18 mentions)
Anti cd44, by BD (15 mentions)
Anti cd105, by BD (11 mentions)
Anti cd34, by BD (11 mentions)
Anti cd45, by BD (11 mentions)
Anti cd73, by BD (10 mentions)
Anti cd31, by BD (9 mentions)
Anti cd106, by BD (4 mentions)
Facscalibur, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Anti hla dr, by BD (3 mentions)
Anti cd166, by BD (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Anti cd45, by Thermo Fisher Scientific (2 mentions)
Anti cd63, by BD (2 mentions)
Anti c kit, by BD (2 mentions)
Anti cd105, by BioLegend (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
70 μm nylon filter, by BD (1 mentions)
Anti ki67, by BD (1 mentions)
28 protocols using anti cd29
1
Isolation and Characterization of fMSCs
2
Flow Cytometry Analysis of TDSCs
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Flow cytometry assay of TDSCs was carried out as described previously [30 (link),31 (link)]. TDSCs (1 × 106) were incubated with 1 µg of AF647-, PE-, PE-Cy7-, or FITC-conjugated specific mouse to rat monoclonal antibodies for 20 min at 4 °C. PE- or FITC-conjugated isotype-matched IgGs (#65209, Abclonal, Wuhan, China) were used as controls. After rinsing with PBS at 300 g for 5 min, the stained cells were resuspended in 400 μL of PBS with 10% FBS (#2128194, XP Biomed Ltd., Shanghai,, China) and analyzed by flow cytometry (Beckman Cytoflex, Beckman Coulter, Brea, CA, USA). Approximately 105 events were counted per sample. The FACSCAN program (Beckman Cytoflex) was used to calculate the percentage of positive signaling cells. The antibodies, including anti-CD29 (#562153, Becton Dickinson, Franklin Lakes, NJ, USA), anti-CD44 (#550974, Becton Dickinson, Franklin Lakes, NJ, USA), anti-CD90 (#551401, Becton Dickinson, Franklin Lakes, NJ, USA), anti-CD34 (sc-7324, Santa Cruz Biotechology, Dallas, TX, USA), and anti-CD31 (FITC-65058, Proteintech, Wuhan, China), were used in this study.
3
Identification and Differentiation of ADSCs
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For identification of ADSCs, ADSC suspensions were incubated with anti-CD29 (APC), anti-CD44 (PC7), anti-CD90 (FITC), anti-CD105 (APC), anti-CD31 (PE), and anti-CD34(APC) antibodies (Becton Dickinson, USA) for 30 min and were analyzed with the FACS Calibur cytometer (Becton Dickinson, USA). Mesenchymal stem cell biomarkers CD29, CD44, CD90, and CD105 were the positive markers of ADSCs. Endothelial marker CD31 and hematopoietic lineage marker CD34 were the negative markers of ADSCs. To identify the differentiation abilities of ADSCs, ADSCs were cultured with adipogenic differentiation medium, osteogenic differentiation medium, and chondrogenic differentiation medium (Cyagen Biosciences, China), according to the manufacturer’s instructions.
4
Characterization of BMSC Surface Markers
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To demonstrate MSCs among BMSCs after primary culture, cell surface CD markers were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA).
Single cell suspensions of BMSCs in phosphate-buffered saline (PBS) were exposed to antibodies directly coupled with a fluorochrome for 30 min on ice. The antibodies used were anti-CD90 (Becton Dickinson) or Mouse IgG1 κ isotype (control; Becton Dickinson) and anti-CD29 (Becton Dickinson) or Hamster IgM κ isotype (control; Becton Dickinson) directly coupled to phycoerythrin and anti-CD90 (Becton Dickinson) or Mouse IgG1 κ isotype (control; Becton Dickinson) coupled to fluorescein-5-isothiocyanate as representative stem cell markers. Similarly, anti-CD45 (eBioscience, San Diego, CA, USA) or Mouse IgG1κ isotype (control; eBioscience) directly coupled to allophycocyanin was used as s representative marker of hematopoietic and endothelial stem cells.
Single cell suspensions of BMSCs in phosphate-buffered saline (PBS) were exposed to antibodies directly coupled with a fluorochrome for 30 min on ice. The antibodies used were anti-CD90 (Becton Dickinson) or Mouse IgG1 κ isotype (control; Becton Dickinson) and anti-CD29 (Becton Dickinson) or Hamster IgM κ isotype (control; Becton Dickinson) directly coupled to phycoerythrin and anti-CD90 (Becton Dickinson) or Mouse IgG1 κ isotype (control; Becton Dickinson) coupled to fluorescein-5-isothiocyanate as representative stem cell markers. Similarly, anti-CD45 (eBioscience, San Diego, CA, USA) or Mouse IgG1κ isotype (control; eBioscience) directly coupled to allophycocyanin was used as s representative marker of hematopoietic and endothelial stem cells.
5
Cytofluorimetric Characterization of EVs
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Cytofluorimetric analysis
was performed using a Guava easyCyte flow cytometer (Millipore, Billerica,
MA, USA) to detect EV fluorescence. In addition, the following PE-conjugated
antibodies were used to characterize EV surface markers: anti-CD81,
anti-CD9, anti-CD107, anti-CD63, anti-CD73, anti-CD44, anti-CD105,
and anti CD29 (all from Becton Dickinson). All experiments were analyzed
with InCyte software.
was performed using a Guava easyCyte flow cytometer (Millipore, Billerica,
MA, USA) to detect EV fluorescence. In addition, the following PE-conjugated
antibodies were used to characterize EV surface markers: anti-CD81,
anti-CD9, anti-CD107, anti-CD63, anti-CD73, anti-CD44, anti-CD105,
and anti CD29 (all from Becton Dickinson). All experiments were analyzed
with InCyte software.
6
Characterization of Rat Bone Marrow Mesenchymal Stem Cells
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Rat BM-MSCs were detached, collected, and centrifuged at 300 × g for 5 min and then suspended in the expansion medium. The viability of BM-MSCS was evaluated in an aliquot using Trypan blue stain by counting the stained live cells and excluding the unstained dead cells as reported by Al-Mutairi et al. [25 ]. For BM-MSC, surface antigen phenotypic analysis and cell markers were assessed [26 (link)]. After being washed twice with phosphate-buffered saline (PBS, PH 7.4; 137-mM NaCl, 2.7-mM KCl, 10-mM Na2HPO4, and 1.8-mM KH2PO4) (Lonza, Germany) containing 1% bovine serum albumin (ALB) (Sigma-Aldrich), 0.2×106 cells were stained with anti-CD34, anti-CD45, anti-CD29, anti-CD73, and anti-CD90 antibodies (BD Biosciences, USA). A FACSCalibur flow cytometer (BD Biosciences) was used to examine conjugated cells as well as determine the proportions of positive and negative populations. A negative sample with untreated isotype cells was used as a control.
7
Isolation and Characterization of BMSCs
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BMSCs were donated by patients who underwent orthopedic surgery after written informed consent. The experiment was approved by the institutional ethics committee of Central South University. Bone marrow aspirate (2 ml) from each donor was inoculated in a 10 cm dish containing medium (DMEM [HyClone, USA], 10% fetal bovine serum [Gibco, USA] and 100 U/ml penicillin G and 100 mg/ml streptomycin [HyClone, USA]) in humidified air at 37 °C. After 3 days of culture, nonadherent cells in the culture supernatant were removed. The BMSCs adhering to the culture dish were cultured in intact medium that was changed every 2 days. When the cultures were confluent, the cells were detached with 0.25% trypsin (Gibco, USA) and then stored or reseeded into culture dishes. The BMSC positive markers including anti-CD90(#555595), anti-CD29(#557332), anti-CD105(#562380), and anti-CD44(#555478), and negative markers, including anti-CD34(#55821) and anti-CD45(#555482), were purchased from BD Biosciences and were used for fluorescence-activated cell sorting (FACS) analysis.
8
Immunophenotyping of Adipose-Derived Mesenchymal Stem Cells
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For immunophenotyping the AdMSCs, the cells were harvested using 0.05% trypsin/EDTA (Invitrogen) at a single cell level and incubated on ice for 30 min with the following monoclonal antibodies: anti-CD29, anti-CD34, anti-CD44, anti-CD45, anti-CD73, and anti-CD90 (BD Pharmingen, San Diego, CA, USA). After washing with phosphate-buffered saline (PBS, Invitrogen), the cells were analyzed using a Cytomics Flow Cytometer (Beckman Coulter, Fullerton, CA, USA).
9
Characterization of Porcine cATMSC-Derived Extracellular Vesicles
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Porcine cATMSC-EV were identified by bead-based flow cytometry, screening for EV and MSC markers. EV were covalently coupled to 4-μm aldehyde/sulphate-latex beads (Invitrogen-ThermoFisher Scientific) with a 15 min incubation, and then blocked for 2 h with BCB buffer (PBS, 0.1% BSA, and 0.01% sodium azide (NaN3); Sigma Aldrich). EV-coupled beads were centrifuged at 2000×g for 10 min and re-suspended in BCB buffer. Next, beads were incubated for 30 min at RT with the fluorochrome-conjugated antibodies anti-CD73-PE and anti-CD90-PE-Cy7 (1:50; both from BD); or the primary Ab anti-CD9 (Clone VJ1/20; 1:10), anti-CD63 (Clone TEA3/18; 1:10), anti-CD81 (Clone 5A6; 1:10), anti-CD29 (1:10; BD), anti-CD44 (1:10; AbD Serotec) or IgG isotype control (1:10; Abcam) followed by incubation with the FITC-goat F(ab')2 anti-mouse IgG (1:10; Bionova) or A488-rabbit anti-rat IgG (1:100; AbD Serotec). EV-coupled beads were washed with BCB buffer and spun down at 2000×g for 10 min after each step. Data was acquired in a FACSVerse flow cytometer (BD), and analysed with FlowJo® v10 (BD).
10
Modulation of Bronchial Epithelial Cell Signaling
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Normal human bronchial epithelial 16HBE14o cells were maintained in MEM plus 10% FCS and 100 U/ml penicillin/streptomycin (GIBCO Invitrogen) and plated in the same medium but without FCS for experimentation. In some experiments cells were pre-treated with inhibitors, namely: AG825 (Calbiochem/Merck Biosciences) (10 μM, 2 h, inhibitor of HER2 activation), anti-CD29 (BD Biosciences) (1 μg/ml, 2 h β1 integrin inhibitor), PF573228 (TOCRIS Bioscience) (3 μM, 1 h, FAK inhibitor), or PNGase F (2 U/ml, 3 h, enzyme that catalyzes the complete removal of N-linked oligosaccharide chains from glycoproteins. Some cells were treated with EPO (4 μg/ml) or MBP(4 μg/ml) for various times.
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