TUNEL staining of OCT-embedded prostate was conducted per manufacturer’s protocol (Roche). Apoptosis analysis of live prostate epithelial cells was conducted by first labeling single cell prostate isolates with CD49f-PE (eBioscience), Sca-1-PE-Cy7 (BioLegend), CD31-eFluor450 (eBioscience), CD45-eFluor450 (eBioscience), Ter119-eFluor450 (eBioscience). This was followed by labeling with Annexin V-APC (BD Pharmingen) and 7-AAD (BD Pharmingen) following the manufacturer’s instructions (BD Biosciences). Data was acquired using a BD FACS Canto (BD Biosciences) and analyzed with FlowJo (v.9.4.10).
Cd31 efluor450
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
CD31-eFluor450 is a fluorochrome-conjugated antibody specific for the CD31 (PECAM-1) cell surface antigen. CD31 is expressed on the surface of endothelial cells, platelets, and some leukocytes. This product can be used for the identification and enumeration of cell populations expressing CD31 in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 efluor450, by Thermo Fisher Scientific (7 mentions)
Ter119 efluor450, by Thermo Fisher Scientific (7 mentions)
Collagenase a, by Roche (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Cd49f pe, by Thermo Fisher Scientific (3 mentions)
Sca 1 pe cy7, by BioLegend (3 mentions)
Facscanto, by BD (3 mentions)
Dispase 1, by Roche (3 mentions)
Dnase 1, by Roche (3 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Sca 1 fitc, by Thermo Fisher Scientific (2 mentions)
Calcium ionophore, by Thermo Fisher Scientific (2 mentions)
Adenosine diphosphate adp, by Merck Group (2 mentions)
Thrombin receptor activator 6 trap 6, by Bio-Techne (2 mentions)
Cd31 fitc, by Thermo Fisher Scientific (2 mentions)
Fitc cd62, by OriGene (2 mentions)
Human vwf, by Prolytix (2 mentions)
Cytofix, by BD (2 mentions)
Ristocetin, by Chrono-Log (2 mentions)
7 aad, by BD (1 mentions)
11 protocols using cd31 efluor450
1
Apoptosis Analysis of Prostate Epithelial Cells
2
BrdU Labeling for Prostate Cell Analysis
3
Isolation of Stromal-Vascular Fraction Cells
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Stromal-vascular fraction single cell suspensions in D-PBS (Life Technologies, Darmstadt, Germany) supplemented with 0.5% BSA and 1 mM EDTA (Sigma-Aldrich) were preincubated with FcBlock [anti-CD16/32 (93, eBioscience), Frankfurt, Germany] for 10 min on ice. Cells were then incubated with Anti-Ter119 MicroBeads (130-049-901, Miltenyi Biotec, Bergisch Gladbach, Germany) on ice for 15 min, to perform erythrocyte depletion by magnetic-activated cell sorting (MACS®) with an OctoMACS Separator according to the manufacturer’s instructions. The flow-through was collected and stained with CD45-FITC (30-F11, eBioscience), CD31-eFluor 450 (390, eBioscience), CD29-PerCP-eFluor 710 (HMb1-1, eBioscience), CD34-Alexa Fluor 647 (RAM34, BD Biosciences, Heidelberg, Germany), Sca-1-Alexa Fluor 700 (D7, eBioscience), and CD140a(Pdgfrα)-biotin (APA5, eBioscience) for 30 min on ice, followed by staining with streptavidin-PE-Cy7 (eBioscience). After antibody staining, samples were washed and sorted with a BD FACS Aria (BD Biosciences). Unstained cells as well as FMO stainings were used as negative controls. Single-stained controls were used for compensation. Data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA). Sorted cells were centrifuged at 300 × g for 5 min for further processing.
4
Isolation and Culture of Satellite Cells
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Satellite cells were isolated by fluorescence-activated cell sorting. Hind limb muscles were minced and digested in Hank's balanced salt solution (HBSS) (Gibco) containing 2 mg ml−1 Collagenase A (Roche), 2.4 U ml−1 Dispase I (Roche), 10 ng ml−1 DNase I (Roche), 0.4 mM CaCl2 and 5 mM MgCl2 for 90 min at 37 °C. Cells were stained with primary antibodies for 30 min on ice. The following antibodies were used: CD31- efluor450 (eBioscience, cat. no. 48-0311, clone: 390, dilution 1:50), CD45-eFluor450 (eBioscience, cat. no. 48-0451, clone: 30-F11, dilution 1:50), Ter119-eFluor450 (eBioscience, cat. no. 48-5921, clone: TER-119, dilution 1:50), Ly-6A/E (Sca1)-FITC (eBioscience, cat. no. 11-5981, clone: D7, dilution 1:50) and α7integrin-647 (AbLab, cat. no. 67-0010-10, clone R2F2, dilution 1:500). Cells were finally washed and suspended in HBSS containing 0.2% w/v BSA and 1% v/v Penicillin–Streptomycin. Flow cytometry analysis and cell sorting were performed on a DAKO-Cytomation MoFlo High Speed Sorter. Cells were plated on gelatin (0,1%, Stem cell Technologies) -coated dishes with GM (20% FBS, 10% horse serum, 1% chick embryo extract in DMEM). After 4–5 days, the medium was replaced with DM (DMEM supplemented with 2% HS and 0.5% chick embryo extract).
5
Fractured Femur Callus Tissue Isolation
6
Isolation of Murine Muscle Satellite Cells
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Tibialis anterior muscles were subjected to enzymatic dissociation in PBS (Cat #14040-091; Gibco) with 2 mg/ml collagenase A (Cat # 10 103 586 001; Roche), 2.4 U/ml Dispase I (Cat # 04 942 078 001; Roche), 10 ng/ml DNase (Cat #11 284 932 001; Sigma-Aldrich), 0.4 mM CaCl 2 , and 5 mM MgCl 2 for 60 min at 37°C under gentle agitation. The supernatants were filtered through a 100-and 40µm cell strainers (#08-771-19, #08-771-1; BD Falcon) and incubated with the following antibodies for 30 min on ice: CD45-eFluor 450 (1/50, #48-0451-82; eBioscience), CD31-eFluor 450 (1/50, #48-0311-82; eBioscience), TER-119-eFluor 450 (1/50, #48-5921-82; eBioscience), Sca1-FITC (1/50, Ly-6A/E FITC, clone D7, #11-5981-82; eBioscience), Itga7-649 (1/500, #67-0010-01; AbLab). MuSCs were isolated as TER-119-/CD45-/CD31-/Itga7+/SCA-1-cells. Isolated satellite cells were used either for RNA extraction and WB analysis, or plated on glass slides (177402; Thermo Fisher Scientific) for immunostaining analysis.
7
Isolation of Fibro-Adipogenic Progenitor Cells
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Hind limb muscles from wild-type mice and Sam68−/− mice were minced and digested in PBS (Sigma-Aldrich) containing 0.1% BSA, 300 μg/ml Collagenase A (Roche), 0.24 U/ml Dispase I (Roche), 2 μg/ml DNase I (Roche), 50 μM CaCl2, and 1 mM MgCl2 for 60 min at 37°C under constant agitation. Digested muscle cells were stained with primary antibodies (1:50) CD31-eFluor450 (eBioscience), CD45-eFluor450 (eBioscience), Ter119-eFluor450 (eBioscience), Sca-1-FITC (BD Pharmingen), and (1:500) α7integrin-APC (AbLab) for 30 min at RT. Cells were finally washed and resuspended in PBS, 0.1% sodium azide, and 0.2% FBS. Flow cytometry was performed with a MoFlo High Speed Cell Sorter (Beckman Coulter) and analysis using FlowJo-10 software. FAP cells were identified as Ter119−/CD45−/CD31−/α7-integrin-/Sca-1+ cells.
8
Prostate Epithelial Cell Quantification
9
Isolation of Fibro-Adipogenic Progenitors from mdx Mice
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FAPs were isolated from C57Bl6 mdx mice at the end of the treatments immediately after the sacrifice. Hind limb muscles for each mouse were minced and put into a 15 ml tube containing 4 ml of digest solution in HBSS (#24020-091,GIBCO) containing 2 mg/ml Collagenase A (#10103586001, Roche), 2.4 U/ml Dispase II (#04942078001, Roche), 10 ng/ml DNase I (#11284932001, Roche) for 90 min at 37°C. Cells were filtered through 100um, 70um and 40um cell strainers (#08-771-19, #08-771-2, #08-771-1, BD Falcon) and resuspended in 0.5 ml of HBSS containing 0.2% w/v BSA and 1% v/v Penicillin-Streptomycin for the staining of cell surface antigens 30 min on ice. The following antibodies were used: CD45-eFluor 450 (1:50, #48-0451-82, eBiosciences), CD31-eFluor 450 (1:50, #48-0311-82, eBioscience), Ter119-eFluor 450 (1:50, #48-5921-82, eBiosciences), Itga7-649 (1:500, #67-0010-01, AbLab) and Sca1-FITC (1:50, 5981-82, eBioscience). Cells were finally washed and resuspended in 1 ml of HBSS containing 0.2% w/v BSA and 1% v/v Penicillin-Streptomycin. FAPs were isolated as Ter119 -/CD45 -/CD31 -/ Itga7 -/Sca1 + cells using a Beckman Coulter MoFlo Legacy high-speed cell sorter.
10
Platelet Function Assays in Blood
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Citrated venous whole blood collected from each participant was used to quantify PAC-1 mAb binding and P-selectin expression, platelet-platelet aggregation, static adhesion on collagen and VWF, and adhesion and thrombus formation under low and high shear flow as previously described (19 (link)). For the clinical investigations, up to 25 mL of peripheral venous blood was obtained; the experimental assay panel used less than 500 μL in all. The activation, aggregation, and static adhesion assays were performed with seven agonists: 100 μM thrombin receptor activator 6 (TRAP-6; Tocris Bioscience, Bristol, UK); 100 μM adenosine diphosphate (ADP; Sigma-Aldrich, St. Louis, MO); 100 μM thromboxane A2 receptor agonist (U46619; Tocris Bioscience); 100 μM epinephrine (Chrono-Log, Havertown, PA); 100 μg/mL collagen related peptide (CRP; R. Farndale, Cambridge University, Cambridge, UK); 100 μM ristocetin (Chrono-Log); and 10 mg/mL calcium ionophore (Invitrogen, Carlsbad, CA). All antibodies were from BD Biosciences (San Jose, CA) with the exception of eFluor450-CD31 (ThermoFisher, Waltham, MA); FITC-CD31 (ThermoFisher); and FITC-CD62 (OriGene Technologies, Rockville, MD). Other material sources include bovine serum albumin, BSA (Sigma-Aldrich); human VWF (Haematologic Technologies, Essex Junction, VT); fibrillary equine type I collagen (Chrono-Log); and cytofix (BD Biosciences).
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