Hearts of mice were minced into 1 mm3 sections and digested with 0.1% collagenase B (Roche Diagnostics GmbH) for 6 min four times in a 37°C water bath (9 (link)). Cell suspensions were obtained by filtering through a cell strainer (40 μm size, BD Falcon) and layered over Ficoll–Hypaque density gradient solution to separate mononuclear cells for flow cytometry. Intracardiac IL-9-producing leukocytes were measured by labeling the harvested cells with the following surface markers: PE anti-mouse CD45, FITC anti-mouse CD4, FITC anti-mouse CD49b, PE-cy7 anti-mouse CD11b, PE-cy7 anti-mouse CD8, or PE anti-mouse Gr-1 antibodies (eBioscience). After washing with PBS, these cells were stimulated with 1 μg/mL ionomycin, 20 ng/mL phorbol myristate acetate (PMA), and 2 μmol/L monensin (eBioscience) for 4 h under 5% CO2 at 37°C in 24-hole culture plates (Costar). After washing, fixing, and permeabilizing according to the manufacturer’s instructions, the cells were stained with APC anti-mouse IL-9 antibody or isotype control antibody. The stained cells were measured and analyzed by FACScalibur flow cytometry (BD Biosciences).
Pe anti mouse cd45
Manufactured by Thermo Fisher Scientific
Sourced in United States
PE anti-mouse CD45 is a flow cytometry reagent that binds to the CD45 cell surface antigen expressed on mouse leukocytes. CD45 is a tyrosine phosphatase that plays a critical role in the activation of lymphocytes. This reagent can be used to identify and analyze mouse leukocyte populations.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Anti mouse cd11b pe cy7, by Thermo Fisher Scientific (2 mentions)
Collagenase a, by Roche (2 mentions)
L glutamine, by Merck Group (2 mentions)
Cell strainer, by BD (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Pe cy7 anti mouse cd8, by Thermo Fisher Scientific (1 mentions)
Collagenase b, by Roche (1 mentions)
Facscalibur flow cytometry, by BD (1 mentions)
Ab88147, by Abcam (1 mentions)
Pe anti mouse cd34, by BD (1 mentions)
Alexa fluor 488 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Facsdiva software, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
5 protocols using pe anti mouse cd45
1
Intracardiac IL-9-Producing Leukocytes Isolation
2
Flow Cytometry Analysis of Aortic Cells
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For flow cytometry analyses, single-cell suspensions of mouse aortae were prepared according to the previous protocol (20 (link)) and stained with following antibodies: 7AAD (00-6993-50, eBioscience, USA), Fluorescent isothiocyanate (FITC) anti-mouse-α-SMA (ab8211, Abcam, United Kingdom), PE-anti-mouse-CD34 (551387, BD, USA), FITC-anti-mouse-CD68 (MA1-82739, Invitrogen, USA), PE-anti-mouse-NK1.1 (ab269324, Abcam, United Kingdom), FITC-anti-mouse-CD19 (11-0193-82, eBioscience, USA), PE-anti-mouse-CD21 (12-0211-82, eBioscience, USA), PE-anti-mouse-CD45 (12-0451-82, eBioscience, USA), and Collagen I (ab88147, Abcam, United Kingdom; at room temperature for 30 min) with Alexa Fluor 488 goat anti-mouse antibody (A32723, Invitrogen, at room temperature for 30 min). Flow cytometry results were analyzed by using FlowJo 7.6 software (FlowJo, LLC., Ashland, Oregon). Statistical analysis was further performed by GraphPad Prism 8.0.
3
Multiparameter Flow Cytometry for IL-1β
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Cells were analyzed on a BD LSR-II flow cytometer equipped with FACSDiva software (both from BD Biosciences, San Jose, CA, USA). FlowJo 7.6.4 software (Tree Star, Ashland, OR, USA) was used for data analysis. The following antibodies were used for cell-surface labeling: PE-anti-mouse CD45, PE-Cy7-anti-mouse CD11b (both from eBioscience, San Diego, CA, USA), APC-Cy7-anti-mouse Ly6C, and PerCP-Cy5.5-anti-mouse Ly6G (both from BD Biosciences). APC-anti-mouse IL-1β/IL-F12 (R&D Systems) was used for the intracellular labeling of IL-1β in fixed and permeabilized (saponin) cells.
4
Retinal Cell Isolation and Immunophenotyping
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Retinas freshly dissected post-euthanasia were collected in neurobasal medium (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 2% B-27 (Thermo Fisher Scientific), and 1% L-glutamine (Merck Life Science). The dissected retinas were mechanically processed with a scalpel. Following gentle resuspension through pipetting for enhanced cell dissociation, 0.2% collagenase A (0.223 U/mg; Roche Diagnostics GmbH, Mannheim, Germany) in Dulbecco’s-modified Eagle’s medium (DMEM) was added and incubated for 30 minutes at 37 °C. Subsequently, cellular suspensions were filtered through a 70-μm CorningTM cell strainer (Thermo Fisher Scientific) and promptly centrifuged for 5 minutes at 600g. The resulting cell pellets were resuspended in complete DMEM medium, and primary fluorescence-labeled antibodies (1:250 anti-mouse CD11b-FITC and 1:500 anti-mouse CD45-PE; eBioscience, Thermo Fisher Scientific) were added. After a 30-minute incubation at 4 °C, two washing steps were performed, and the cells were ultimately analyzed using a FACS Canto flow cytometer (Becton Dickinson, Franklin Lakes, NJ, United States). Flow cytometry data were analyzed with FlowJo software (FlowJo LLC, Ashland, OR, United States) at the Tissue Culture Facility (ACTI, University of Murcia and IMIB).
5
Retinal Cell Isolation and Flow Cytometry
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Retinas were collected in neurobasal medium (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 2% B-27 (Thermo Fisher Scientific) and 1% L-glutamine (Merck Life Science) after CO2 euthanasia and dissected mechanically with scalpel. After resuspending up and down twice with micropipette to improve cell dissociation, 0.2% collagenase A (0.223 U/mg; Roche Diagnostics GmbH, Mannheim, Germany) in DMEM was added and incubated for 30 min at 37 °C. After that, cellular suspensions were filtered through a 70-μm Corning™ cell strainer (Thermo Fisher Scientific) and immediately centrifuged for 5 min at 600g. Cell pellets were resuspended in complete DMEM medium and primary fluorescence-labelled antibodies were added (1:250 anti-mouse CD11b-FITC; eBioscience, Thermo Fisher Scientific; and 1:500 anti-mouse CD45-PE; eBioscience, Thermo Fisher Scientific). After incubation for 30 min at 4 °C, two washing steps were performed and finally acquired in a FACS Canto flow cytometer. Flow cytometry data were analysed with FlowJo software (FlowJo LLC, Ashland, OR, USA) at the Tissue Culture Facility (ACTI, University of Murcia and IMIB-Arrixaca).
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