Collected MSCs were incubated for 30 min at room temperature with the following specific antibodies: PE mouse anti-human CD29, FITC rat anti-human CD44, FITC mouse anti-human CD105, FITC rat anti-human CD45, APC mouse anti-human CD34, and PE mouse anti-human HLA-DR (all from BD Pharmingen). As a control, the cells were stained with the appropriate isotype antibodies. At the end of co-culture, the CD4+ T cell apoptosis was analyzed by using an Annexin V-PE Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. To detect Treg cells, a Human Regulatory T Cell Staining Kit (eBioscience) containing an anti-CD4-FITC/CD25-APC cocktail and anti-Foxp3-PE was used according to the manufacturer’s instruction. In addition, we used a Human Th1/Th2/Th17 Phenotyping kit (BD Pharmingen) to analyze the T helper cell subsets. All samples were analyzed using a BD Biosciences Influx cell sorter.
Pe mouse anti human cd29
Manufactured by BD
Sourced in Germany
The PE mouse anti-human CD29 is a laboratory reagent used for the detection and identification of CD29 (also known as Integrin β1) on the surface of human cells. CD29 is a cell surface marker that plays a role in cell adhesion and signaling. This product can be used in flow cytometry and other immunoassays to analyze the expression of CD29 on various cell types.
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Lab products found in correlation
Pe mouse anti human hla dr, by BD (4 mentions)
Fitc mouse anti human cd105, by BD (3 mentions)
Pe mouse anti human cd73, by BD (3 mentions)
Pe mouse anti human cd90, by BD (2 mentions)
Fitc mouse anti human cd45, by BD (2 mentions)
Fitc mouse anti human hla abc, by BD (2 mentions)
Fitc mouse anti human cd31, by BD (2 mentions)
Fitc mouse anti human cd44, by BD (2 mentions)
Apc mouse anti human cd34, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Human regulatory t cell staining kit, by Thermo Fisher Scientific (1 mentions)
Anti cd4 fitc cd25 apc cocktail, by Thermo Fisher Scientific (1 mentions)
Influx cell sorter, by BD (1 mentions)
Human th1 th2 th17 phenotyping kit, by BD (1 mentions)
Pe mouse anti human cd166, by BD (1 mentions)
Calibur flow cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Human bone marrow derived mscs, by Lonza (1 mentions)
Fcs express, by De Novo Software (1 mentions)
Fitc mouse anti human cd61, by BD (1 mentions)
6 protocols using pe mouse anti human cd29
1
Phenotypic Analysis of Immune Cell Subsets
2
Characterization of Mesenchymal Stem Cells
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H9‐derived MSCs and human bone marrow‐derived MSCs (Lonza, Walkersville, MD,
http://www.lonza.com ) were grown to confluence, harvested by using 0.25% trypsin/EDTA, and resuspended in buffer containing phosphate‐buffered saline (PBS), 2% HEPES buffer, 2% FBS, and 0.1% bovine serum albumin (BSA) as previously described 43 . Cells (1 × 106) were incubated with phycoerythrin (PE) mouse anti‐human CD90, PE mouse anti‐human CD73, fluorescein isothiocyanate (FITC) mouse anti‐human CD44, FITC mouse anti‐human CD45, FITC mouse anti‐human HLA‐ABC, PE mouse anti‐human CD29, PE mouse anti‐human CD166, PE mouse anti‐human HLA‐DR, FITC mouse anti‐human CD105, or FITC mouse anti‐human CD31 (BD Biosciences, San Jose, CA,
http://www.bdbiosciences.com ). Nonspecific fluorescence was determined by using isotype‐matched monoclonal antibodies. A total of 10,000 events were collected on a BD fluorescence‐activated cell sorting Calibur Flow Cytometer instrument by using CellQuest software (BD Biosciences). Analyses of results and corresponding graphs were generated by using FlowJo software (Tree Star, Ashland, OR,
http://www.flowjo.com ) 43 44 45 .
3
Flow Cytometry Analysis of CD61 and CD29
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Cells, treated with CT20p, were washed with 5% FBS in PBS and stained with FITC mouse anti-human CD61, PE mouse anti-human CD29 (BD Bioscience), or a corresponding isotype control antibody (BD Bioscience). Data were acquired with an Accuri C6 flow cytometer and analyzed with FCS Express (Denovo, Niwot, CO, USA) software.
4
Flow Cytometric Characterization of Cells
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Cells (5 × 105) were suspended in 100 μL PBA buffer (PBS, 0.1% NaN3, 0.1% BSA). Appropriate antibodies, APC mouse anti-human CD133 (Miltenyi Biotec, Germany); PE-Cy7 mouse anti-human CD44, PE mouse anti-human CD29, FITC-mouse anti-human CD49b (BD Biosciences, San Jose, CA, USA) were added to each cell sample and incubated in the dark for 20 min. A further 100 μL PBA was added; cells were pelleted to remove unbound antibody and re-suspended in 200 μL PBA buffer. The samples were then acquired on a Dako CyAn ADP flow cytometer (Beckman Coulter, Brea, CA, USA).
5
Flow Cytometry Immunophenotyping of Cells
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Cells were digested by trypsinization, then harvested by centrifugation at 1500 rpm for 5 min, washed in ice-cold phosphate-buffered saline (PBS), and finally resuspended at a ratio of 105 cells/antibody. Cells were then incubated for 30 min. on ice in the dark with the appropriate isotype controls or the preconjugated antibodies. Next, cells were washed and resuspended in PBS (500 μL) and then analyzed in the Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) following the method described by Elsafadi et al. (40). The following antibodies were used: APC Mouse Anti-Human CD44, FITC-PE-APC-mouse-IgG1k-isotype-control, FITC Mouse Anti-Human CD45, FITC Mouse Anti-Human CD31, PE-mouse-anti-human-CD73, PE Mouse Anti-Human CD29, and PE Mouse Anti-human HL-ADR (all available from BD Biosciences).
6
Phenotypic Characterization of NHAC-kn Cells
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NHAC-kn cells (passage 5) were grown to near confluence, harvested by 0.25% trypsin/EDTA, washed with PBS and re-suspended in staining solution consisting of 2% FBS and 2% HEPES in PBS. Cell suspensions (1 × 106 cells) were mixed with PE mouse anti-human CD90 (BD Pharmingen), PE mouse anti-human CD73 (BD Pharmingen), PE mouse anti-human CD29 (BD Pharmingen), PE mouse anti-human HLA-DR (BD Pharmingen), FITC mouse anti-human CD44 (BD Pharmingen), FITC mouse anti-human HLA-ABC (BD Pharmingen), FITC mouse anti-human CD105 (BD Pharmingen), FITC mouse anti-human CD45 (BD Pharmingen), and FITC mouse anti-human CD31 (BD Pharmingen).28 (link) Nonspecific fluorescence was determined by incubation of cell aliquots with isotype-matched monoclonal antibodies (IgG1-PE and IgG2b-FITC). Samples were run on a Becton–Dickinson LSR II Flow Cytometer (BD Biosciences) instrument using FACS Diva software (Becton Dickinson). For each analysis, a minimum of 10,000 cells was assayed. Data was analyzed using FloJo Software (Tree Star, Inc.).
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