M-chlorin e6 PDT was performed as described above. Mice were euthanized by cervical dislocation on day 2 after LED irradiation. The tumors were removed and treated with 10% collagenase (Wako), shredded using a gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), and passed through a 70-μm filter. Next, 1.0 × 106 cells were mixed with 1 μL of Fc-block/100 μL of stain buffer (BD Biosciences, San Jose, CA) and left on ice for 10 min. Subsequently, 1 μL of fluorescent antibody (CD11b-APC [BD Biosciences], CD206-BV421 [Biolegend, San Diego, CA], CD80-PE [BD Biosciences], CD86-PE-Cy7 [BD Biosciences], CD45-PE-Vio®770 [Miltenyi Biotec], CD3-FITC [Miltenyi Biotec], CD8a-APC-H7 [BD PharMingen, San Diego, CA], CD127-APC [BD PharMingen], CD25-PE [BD Biosciences], or CD4-BV421 [Biolegend]) diluted with 100 μL of stain buffer was added and left on ice for 30 min. The cells were then washed with 500 μL of phosphate-buffered saline (PBS) and suspended in 500 μL of stain buffer. Next, 5 μL of 7-AAD (BD Biosciences) was added to prepare a sampling solution, which was analyzed on a BD FACSVerse™ (BD Biosciences).
Cd45 pe vio770
Manufactured by Miltenyi Biotec
Sourced in Germany
The CD45-PE-Vio770 is a fluorochrome-conjugated monoclonal antibody that binds to the CD45 antigen, which is expressed on the surface of most human leukocytes. It can be used for the identification and enumeration of leukocyte subsets in flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cd73 pe, by Miltenyi Biotec (6 mentions)
Cytoflex, by Beckman Coulter (4 mentions)
Cd31 percpvio700, by Miltenyi Biotec (4 mentions)
Cd90 fitc, by Miltenyi Biotec (4 mentions)
Cd34 fitc, by Miltenyi Biotec (3 mentions)
Cd44 percp 44pp2, by Immunostep (2 mentions)
Cd105 percp vio700, by Miltenyi Biotec (2 mentions)
Cd44 pe vio770, by Miltenyi Biotec (2 mentions)
7 aad, by BD (1 mentions)
Cd3 fitc, by Miltenyi Biotec (1 mentions)
Cd8a apc h7, by BD (1 mentions)
Cd127 apc, by BD (1 mentions)
Cd206 bv421, by BioLegend (1 mentions)
Fc block, by BD (1 mentions)
Cd86 pe cy7, by BD (1 mentions)
Cd25 pe, by BD (1 mentions)
Cd80 pe, by BD (1 mentions)
Cd4 bv421, by BioLegend (1 mentions)
Cd11b apc, by BD (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
8 protocols using cd45 pe vio770
1
Tumor-Infiltrating Immune Cell Analysis
2
Multiparametric Flow Cytometry for Cell Phenotyping
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Flow cytometry was performed with CytoFlex (Beckman Coulter, Fullerton, California) flow cytometer (minimum 30 000 events), with CD90‐FITC (REA897), CD73‐PE (REA804), CD34‐FITC (AC136), CD45‐PEVIO770 (REA747) and CD31‐PERCPVIO700 (REA730) (Miltenyi, Bergisch Gladbach, Germany), and CD44‐PERCP (44PP2) (Immunostep, Salamanca, Spain) antibodies.25
3
Immunophenotype Analysis of ASCs and FLSs
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Immunophenotype analysis was performed on three different representative populations for each cell type. For ASCs, the experiments were conducted at passage 3 and cells incubated for 10 min at 4 °C in the dark with anti-human antibodies: CD90-FITC, CD44-PE CD73-PE, CD105-PerCP-Vio700, CD34-PE-Vio770, and CD45-PE-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany). For FLSs, analyses were conducted at passages 0 and 1, and cells stained for 10 min at 4 °C in the dark with anti-human antibodies: CD73-PE (Miltenyi) and CD14-FITC (Ancell Corporation, Bayport, MN, USA). Unstained samples for each population were used as negative controls, and data were acquired by the CytoFLEX flow cytometer (Beckman Coulter, CA, USA) collecting a minimum of 50,000 events.
4
Multiparameter Flow Cytometry of ASCs
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ASCs, with and without 50% OA-SF, were analyzed by flow cytometry with a CytoFLEX flow cytometer (Beckman Coulter, Fullerton, CA, USA), collecting at least 50,000 events. Antibodies: anti-CD90-FITC (REA897, Miltenyi Biotec, Bergisch Gladbach, Germany), CD73-PE (REA804, Miltenyi), CD105-PerCP-Cy5.5 (43A3, BioLegend, San Diego, CA, USA), CD44-PE-Vio770 (REA690, Miltenyi), CD34-FITC (AC136, Miltenyi), CD271-PE (REA844, Miltenyi), CD31-PerCP-Vio700 (REA730, Miltenyi) and CD45-PE-Vio770 (REA747, Miltenyi). Aggregates were removed from gating events on a FSC-H and FSC-A plot.
5
Phenotypic Characterization of ASCs
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A CytoFlex (Beckman Coulter, Fullerton, CA, USA) flow cytometer, collecting a minimum of 30,000 events, was used to confirm ASCs phenotype [31 (link)]. The following antibodies were used: CD90-FITC (REA897), CD73-PE (REA804), CD34-FITC (AC136), CD45-PEVIO770 (REA747) and CD31-PERCPVIO700 (REA730) (Miltenyi, Bergisch Gladbach, Germany) and CD44-PERCP (44PP2) (Immunostep, Salamanca, Spain). Incubation was allowed for 30 min in the dark at 4 °C, as per the manufacturer’s instructions.
6
Phenotypic analysis of hMSCs
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hMSCs were incubated for 30 min at 4 °C in the dark with anti-human antibodies: CD90-FITC, CD73-PE, CD105-PerCP-Vio700, CD45-PE-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany). Unstained samples were used as negative controls, and data were acquired with a CytoFLEX flow cytometer (Beckman Coulter, CA, USA) collecting a minimum of 30,000 events.
7
Hematopoietic Stem Cell Immunophenotyping
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On day 19 of cultures, the methylcellulose cultures were washed with PBS. Living cells were counted in trypan blue and stained with the following antibodies in PBS 4%BSA at 4 °C 45 min: CD45Pe-Vio770 (Miltenyi), CD34Vioblue (Miltenyi), CD41aPE (BD), CD43FITC (Miltenyi), CD31FITC (Beckman Coulter), CD235aAPC (BD), IL1-RAP APC (R&D), CD38PE (Miltenyi), CD71PE (BD), CD14FITC (Beckman Coulter), CD33APC (BD), CD133APC (BD), BB9PE (BD), SSEA1PE (BD). After 1 h, cells were washed and resuspended in PBS 4%BSA with viability staining reagent 1 µg/mL (7-AAD) 7-aminoactinomycin D (Sigma Aldrich). Stained cells were analyzed with a MACSQuant 10 (Miltenyi Biotec) flow cytometer and Flowjo analysis software.
8
Flow Cytometry Phenotyping of Cells
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Flow cytometry analysis was performed with following antibodies: CD90-FITC (REA897), CD73-PE (REA804), CD44-PEVIO770 (REA690), CD34-PE (AC136), CD45-PEVIO770 (REA747) and CD31-PERCPVIO700 (REA730) (Miltenyi, Bergisch Gladbach, Germany). Staining was performed at 4 °C for 30 min in the dark following dilution suggested by manufacturer. A CytoFlex (Beckman Coulter, Fullerton, CA, USA) flow cytometer was used collecting a minimum of 30,000 events.
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