Cell surface staining was conducted with the appropriate fluorochrome-conjugated Abs for 30 min at 4°C. The following specific monoclonal antibodies (mAbs) were used: antigen-presenting cells (APC)-cy7-conjugated anti-mouse CD45, FITC-conjugated anti-mouse CD3, Percy5.5-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD8, PEcy7-conjugated anti-mouse NK1.1, BV421-conjugated anti-mouse Nkp46, BV421-conjugated anti-mouse major histocompatibility complex II (MHC-II), APC-conjugated anti-mouse F4/80, PE-conjugated anti-mouse CD11c, PE-CF594-conjugated anti-mouse CD11b and APC-conjugated anti-mouse CD27 (all purchased from BD Bioscience). Flow cytometric (FCM) analyses were performed on a CyAn ADP analyzer (Beckman Coulter, Inc., Kraemer Boulevard Brea, CA, USA), with data analyzed using FlowJo Version 6.1 software (TreeStar, Asland, OR, USA).
Apc cy7 conjugated anti mouse cd45
Manufactured by BD
The APC-CY7 conjugated anti-mouse CD45 is a flow cytometry reagent that binds to the CD45 surface marker expressed on mouse leukocytes. It is used for the identification and enumeration of various immune cell populations in mouse samples.
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Lab products found in correlation
Cyan adp analyzer, by Beckman Coulter (1 mentions)
Fitc conjugated anti mouse cd3, by BD (1 mentions)
Fitc conjugated anti mouse cd8, by BD (1 mentions)
Pecy7 conjugated anti mouse nk1, by BD (1 mentions)
Apc conjugated anti mouse f4 80, by BD (1 mentions)
Fitc conjugated anti mouse cd4, by BD (1 mentions)
Apc conjugated anti mouse cd11b, by BD (1 mentions)
Pe conjugated anti mouse f4 80, by BD (1 mentions)
Apc conjugated anti mouse gr1, by BioLegend (1 mentions)
Fitc conjugated anti mouse cd8a, by BioLegend (1 mentions)
Fitc conjugated anti mouse cd11b, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Anti mouse cd16 32 antibody clone 93, by BioLegend (1 mentions)
Pe conjugated anti mouse cd4, by BD (1 mentions)
Cell activation cocktail with brefeldin a, by BioLegend (1 mentions)
Papain, by Worthington (1 mentions)
4 protocols using apc cy7 conjugated anti mouse cd45
1
Comprehensive Immune Cell Profiling
2
Lymph Node Immune Cell Analysis
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On day 14 post-surgery, single-cell suspension was prepared from draining submandibular and cervical lymph nodes. The expression of CD4+ T cells and macrophages in the peripheral lymph nodes were analyzed using flow cytometry. Upon resuspension in binding buffer at a density of 2 × 107 cells/ml, the cells were then stained with APC-CY7 conjugated anti-mouse CD45 (#561037, BD Biosciences), AF700 conjugated anti-mouse CD3 (#557984, BD Biosciences) and FITC conjugated anti-mouse CD4 (#561831, BD Biosciences) to label the surface markers of CD4+ T cells. The surface markers of macrophages were detected by APC-conjugated anti-mouse CD11B (#561039, BD Biosciences) and PE-conjugated anti-mouse F4/80 (#565410, BD Biosciences).
3
Flow Cytometry Immunophenotyping Assay
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Single-cell suspensions were incubated with anti-mouse CD16/32 antibody (clone 93; Biolegend, San Diego, CA) for 5 minutes prior to staining for immune cell markers for 15 minutes at room temperature. The following monoclonal antibodies were used: APC-CY7 conjugated anti-mouse CD45 (BD Biosciences Cat:557659 Clone:30-F11), FITC conjugated anti-mouse CD11b (Biolegend Cat:101205 Clone:M1/70), APC conjugated anti-mouse Gr1(Biolegend Cat:108412,Clone: RB6-8C5), FITC conjugated anti-mouse CD8a(Biolegend Cat:100706 Clone:53-6.7), PE conjugated anti-mouseCD4(BD Biosciences Cat:553048 Clone: RM4-5), APC conjugated anti-mouse PD-L1(Biolegend Cat:124311 Clone: 10F.9G2), and IFN-γ (XMG1.2).The flow cytometry analyses were performed using a BD Fortessa Flow Cytometer (BD Fortessa). BD FACS Diva software V.5.0.1 (BD) or Flow Jo (Tree Star) was used for data processed. For cytokine staining, harvested cells were incubated in RPMI-1640 medium with cell activation cocktail with brefeldin A (Bio legend) for 6 hours at 37°C, and stimulated cells were stained as described above.
4
Enriching Endothelial Cells from Mouse Brain
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The procedures of enriching pCECs were adapted from a published protocol40 (link). Briefly, animals were perfused transcardially with ice-cold PBS under anesthesia. Brain tissue was collected, washed with HEPES-MEM buffer once, and digested with DNase (Cat. #LK03172, Worthington, NJ) and papain (Cat. #LK003178, Worthington, NJ) for 70 minutes. Then samples were mixed with 30% BSA and centrifuged at 1290 g for 10 min. Myelin was removed from the top of tubes and cells were suspended in 10% FBS-DMEM medium. Cells were then purified with mouse CD31 magnetic beads (Cat. #130-097-419, Miltenyi, Germany) following the manufacturer’s instructions. To verify the cell purity, a portion of the cells were incubated with magnetic beads, APC-Cy7-conjugated anti-mouse CD45 (Cat. #557659, BD Bioscience, CA), and PE-conjugated anti-mouse CD31 (Cat. #553373, BD Bioscience, CA) followed by cell sorting procedures and analyzed by flow cytometry (BD, LSRFortessa).
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