Kidney T cells were isolated using FACS. Briefly, single-cell suspension of KMNCs was preincubated with anti-CD16/CD32 Fc block (clone S17011E, BioLegend, 156604) stained in Cell Staining Buffer (BioLegend) with fluorochrome-labeled antibodies: APC-Cy7 anti-CD45 (clone 30-F11, BioLegend, 103116) and BV421 anti-TCRβ (clone H57-957, BioLegend, 109230). Live Dead Aqua (Thermo Fisher Scientific) was stained for viability assay. Live Dead Aqua–CD45+TCRβ+ cells were sorted with FACSAria II Cell Sorter (BD Biosciences). Splenic T cells were isolated from single-cell suspension of spleens using a T Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s guidelines.
Anti cd45 apc cy7 clone 30 f11
Manufactured by BioLegend
Sourced in United States
Anti-CD45 APC/Cy7 (clone 30-F11) is a fluorescently labeled antibody that binds to the CD45 protein, which is expressed on the surface of hematopoietic cells. This product can be used for the identification and analysis of CD45-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Phosflow kit, by BD (2 mentions)
Anti stat3 y705 pe clone 4 p stat3, by BD (2 mentions)
Anti stat3 s727 pe clone 49 p stat3, by BD (2 mentions)
Anti pjak2 y1007 1008 alexa fluor 647 clone e132, by Abcam (2 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Live dead aqua, by BD (1 mentions)
T cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Anti cd16 32, by BioLegend (1 mentions)
Recombinant rankl, by Oriental Yeast (1 mentions)
Biotinylated uea 1, by Vector Laboratories (1 mentions)
Anti 1 a 1 e fitc clone m5 114.152, by BioLegend (1 mentions)
Anti s100a8, by Thermo Fisher Scientific (1 mentions)
Anti cd11b clone m1 70, by BD (1 mentions)
Anti gr 1 clone rb6 8c5, by BD (1 mentions)
Zombie violet, by BioLegend (1 mentions)
Anti epcam pe, by BioLegend (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
6 protocols using anti cd45 apc cy7 clone 30 f11
1
Isolation of Kidney and Splenic T Cells
2
Murine Immune Cell Profiling
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The following fluorescent anti–mouse antibodies were used for flow cytometry: anti-CD16/32, APCCy7-anti-CD45 (clone 30 F-11), APCCy7- or PECy7-anti-TER119 (clone TER-119), PE- or PECy7-anti-EpCAM (CD326, clone G8.8), FITC- or PECy7-anti-MHCII (I-A/I-E, clone M5/114.15.2), APC-anti-Ly51 (clone 6C3; BioLegend), PE-anti-CD80 (clone 16-10A1), PE-anti-CD24 (clone M1/69; eBioscience), and biotinylated UEA-1 (Vector Laboratories) antibodies. An agonistic anti-LtbR antibody was purchased from Alexis Biochemicals. Recombinant RANKL was a generous gift from Oriental Yeast Co. Anti-RANKL antibodies were prepared from a subclone of hybridoma obtained by fusing mouse myeloma cells with B cells.
3
Multiparameter Flow Cytometry for Immune Cell Analysis
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Cell suspensions were surface labeled with fluorochrome-conjugated antibodies for 10 m at 4 °C in staining buffer (PBS containing 1% BSA). For phosphoprotein staining, cells were fixed and permeabilized using the Phosflow kit (BD) according to manufacturer’s protocol before staining for 1 h at room temperature. Data were acquired using LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star). The following antibodies were used for flow cytometry analysis: anti-CD45 APC/Cy7 (clone 30-F11; BioLegend), anti-Ly6G PE/ biotin (clone 1A8; BD, BioLegend), anti-CD11c PerCP (clone N418; BioLegend), anti-F4/80 PE (clone BM8; eBioscience), anti-I-A/I-E FITC (clone M5/114.152; BioLegend), anti-Gr1 (clone RB6–8C5; BD Pharmingen), anti-CD11b (clone M1/70; BD Pharmingen), anti-STAT3 (Y705) PE (clone 4/P-STAT3, BD Biosciences), anti-STAT3 (S727) PE (clone 49/P-STAT3, BD Biosciences), anti-pJAK2 (Y1007,1008) Alexa Fluor 647 (clone E132, abcam), anti-S100a8 (catalog PA5-86063, ThermoFisher Scientific).
4
Multicolor Flow Cytometry of Tissue Cells
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Cell pellets from tissue dissociation were resuspended in 100 μl of FACS stain buffer (2% FBS in PBS) with 1 μl of anti–CD31–Brilliant Violet 786 (clone 390, BD Biosciences), 1 μl of anti–CD45–APC/Cy7 (clone 30-F11, BioLegend), 1 μl of parenchymal cell marker [anti–GLUT2-PE (Bioss), anti–EPCAM-PE (BioLegend), or anti–cTnT-PE (Miltenyi Biotec)], 0.02 μl of Alexa Fluor 647–conjugated AxV, 5 ml Zombie Violet (BioLegend), and 0.1 μl of CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific). Cells were stained on ice for 45 min away from the light and then centrifuged at 200g for 5 min at 4°C, and fluorescence in different cell types was analyzed on an Attune NxT flow cytometer (Thermo Fisher Scientific).
5
Multicolor Flow Cytometry for Myeloid Cell Profiling
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Cells counts were performed using a Z2 Coulter Counter (Beckman Coulter, USA). 3X106 cells from single cell suspensions were incubated at 4 °C for 20 min with the following fluorochrome-conjugated anti-mouse markers: anti-CD45 APC-Cy7 (clone 30-F11; Biolegend, San Diego, USA), anti-CD11b PE-Cy7 (clone M1/70; BD Biosciences), anti-CD11c Pacific Blue (clone N418; Biolegend), anti-Ly6C FITC (clone HK1.4; Biolegend), anti-Ly6G Alexa Fluor 647 (clone 1A8; Biolegend), anti-F4/80 APC (clone BM8; eBioscience), and anti-CD206 (mannose receptor; MR) Alexa Fluor 488 (clone C068C2; Biolegend). Fc receptor block (anti-CD16/32 antibody) was added to all markers cocktails. Intracellular CD206 labelling was performed using a CytoFix/CytoPerm kit (BD Biosciences, USA). After surface receptor labelling, cells were permeabilized and incubated with the marker for 30 min at 4 °C in the dark before being washed twice in 1× Perm/Wash buffer (BD Biosciences) and resuspend in FACS buffer. A BD FACS Canto II flow cytometer (BD Biosciences) was used to acquire data. Data was analysed using FlowLogic FCS analysis software (Inivai Technologies, Melbourne, Australia).
6
Multiparameter Flow Cytometry of Signaling
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Cell suspensions were surface labelled with fluorochrome-conjugated antibodies for 10 minutes at 4°C in staining buffer (PBS containing 1% BSA). For phosphoprotein staining, cells were fixed and permeabilized using the Phosflow kit (BD) according to manufacturer’s protocol before staining for 1 hour at room temperature. Data were acquired using LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star). The following antibodies were used for flow cytometry analysis: anti-CD45 APC/Cy7 (clone 30-F11; BioLegend), anti-Ly6G PE/biotin (clone 1A8; BD, BioLegend), anti-STAT3 (Y705) PE (clone 4/P-STAT3, BD Biosciences), anti-STAT3 (S727) PE (clone 49/P-STAT3, BD Biosciences), anti-pJAK2 (Y1007,1008) Alexa Fluor 647 (clone E132, abcam), anti-Erk1/2 (pT202/Yp204) PE (612593, BD Biosciences), anti-pIKKα/β (S176/180) PE (14938S, Cell Signaling Technology), anti-p-p38 (T180/Y182) FITC (612594, BD Biosciences), anti-S100a8 (clone E4F8V; Cell Signaling Technology), anti-S100a9 (clone D3U8M; Cell Signaling Technology), anti-pSHP2 (Y580) PE (MA5-28045, Invitrogen), goat anti-rabbit IgG (H+L) secondary antibody FITC (Invitrogen).
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