EAE mice receiving 2D2-Kv1.3 KO or 2D2-WT Th cells were anesthetized, perfused with HBSS, and blood, lymph nodes, spleens and brains were removed. CNS tissue was digested and enriched for mononuclear cells. Single cell suspensions were made from blood, lymph nodes, and spleens as described below. Collected cells were stimulated in media containing PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO), ionomycin (1 μg/ml; Sigma-Aldrich), and monensin (4 μl/6 ml; GolgiStop, BD Biosciences) at 37°C for 4-6 hrs. Cells were washed with FACS buffer (2% FBS in PBS) and surface stained with anti-CD4 (BD Biosciences, RM4-5) Percep or APC, anti-CD45.2 FITC, anti-CD11b APC, and anti-Ly6G PE(BD Bioscience, San Jose, CA and eBioscience, San Diego, CA). Flow cytometry was performed using a FACS Calibur flow cytometer and data were analyzed using FlowJo software (Treestar, Ashland, OR).
Anti ly6g pe
Manufactured by BD
Sourced in United States
Anti-Ly6G-PE is a fluorochrome-conjugated antibody used for the detection and quantification of Ly6G-expressing cells in flow cytometry applications. Ly6G is a marker specific for mature neutrophils in mice.
Automatically generated - may contain errors
Lab products found in correlation
Anti ly6c fitc, by BD (6 mentions)
Anti cd11b apc, by BD (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Anti ly6c apc, by BD (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Nigericin, by Merck Group (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti cd11b bv421, by BD (2 mentions)
Anti f4 80 efluor450, by Thermo Fisher Scientific (2 mentions)
Anti cd11b percp cy5, by BD (2 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Anti cd4, by BD (1 mentions)
Facscalibur flow cytometer, by Tree Star (1 mentions)
Anti cd45.2 fitc, by BD (1 mentions)
Anti cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd4 percp, by BD (1 mentions)
Anti cd25 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd11b pacific blue, by Thermo Fisher Scientific (1 mentions)
17 protocols using anti ly6g pe
1
Multimodal Immune Profiling in EAE Mice
2
Immune Cell Profiling in Atherosclerosis
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Splenocytes and blood were isolated from apoE-/-, catK-/-//apoE-/-, wt, and catK-/- mice. Erythrocytes in peripheral blood and spleen were removed by hypotonic lysis with NH4Cl. Cells were incubated first with anti-CD16/32 (eBioscience, San Diego, CA) to block Fc receptor binding to antibodies on macrophages, neutrophils and mast cells and stained with anti-CD3-FITC, anti-CD8-Pacific blue, anti-CD25-APC, anti-CD45R(B220)-Pe-Cy7 (eBioscience, San Diego, CA) and anti-CD4-PerCp (BD-Biosciences Pharmingen, San Diego, CA). Foxp3-positive cells were detected with PE anti-mouse/rat Foxp3 Staining Set, according to the manufacturer’s instruction (eBioscience, USA). Peripheral blood leukocytes were incubated with anti-CD11b-pacific blue (eBioscience, San Diego, CA) and anti-ly6G-PE (BD-Biosciences Pharmingen, San Diego, CA) to detect monocytes (CD11b+ly6G-) and granulocytes (CD11b+ly6G+). For measurement of inflammatory monocytes (CD11b+Ly6G-Ly6Chigh) cells were stained with anti-Ly6C-FITC (Miltenyi Biotec). Blood natural killer (NK) cells were stained by anti-CD49b(DX5)-APC (Miltenyi Biotec).
3
Delayed-Type Hypersensitivity Evaluation
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Seven days after immunization with OVA as above, mice were challenged s.c. at right footpad with 200 μg of OVA in 20 μl PBS. As a control, the same volume of PBS was injected into left footpad. Footpad thickness was measured with a dial vernier caliper (Teclock) on day 1 and 2. The magnitude of the DTH response was calculated as follows; footpad swelling (μm) = thickness of OVA-injected footpad − thickness of PBS-injected footpad. For histological analysis of the DTH lesions, paws were removed on day 2 and fixed with 10%-formaldehyde neutral buffer solution (Nacalai). After decalcification by a standard protocol, specimens were embedded in paraffin and were stained with hematoxylin-eosin (H&E). For analysis of the tissue-infiltrating cells, paws were thoroughly minced with scissors and then were incubated at 37°C for 1 hour in Hank's solution containing 1.0 mg/ml collagenase II (Worthington), 1.0 mg/ml dispase (Sigma-Aldrich) and 40 μg/ml Dnase I (Roche). After removing debris with 70 μm cell-strainers, cells were re-suspended into 33.7% Percoll (GE Healthcare) and pelleted by centrifugation at 1,000 g, for 20 minutes at 24°C. Cells were stained with anti-CD45.2-APC (eBioscience), anti-CD11b-FITC (eBioscience), anti-Ly6G-PE (BD PharMingen), anti-CD3ε-APC (eBioscience) antibodies, and analyzed by flow cytometry for expression of respective molecules.
4
Inflammasome Activation Pathway Analysis
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PMA, ATP, nigericin, PF-431396, 4′,6-diamidino-2-phenylindole (DAPI), and the anti-Flag M2 antibody were purchased from Sigma. R406, PF-562271, and Z-VAD-FMK were purchased from Selleckchem. Anti-Pyk2¸ anti-Syk, anti-p-Pyk2, anti-p-FAK, anti-p-Syk, and anti-GAPDH were purchased from Cell Signaling. Anti-FAK, anti-ASC, anti-caspase-1, and anti-IL-1β were purchased from Santa Cruz. Anti-Ly6G-PE and anti-CD11b-APC were purchased from BD Bioscience. MSU, PF-573228, anti-NLRP3, anti-mCherry, anti-F4/80-FITC, and Alexa Fluor 488–conjugated goat anti-rabbit IgG were purchased from InvivoGen, Tocris Bioscience, AdipoGen, Abcam, eBioscience, and Invitrogen, respectively. Plasmids encoding His-tagged wild-type and mutant (Y146F) ASC were generated by ligation of amplified DNA fragments to NdeI/XhoI-treated pET15b (Novagen, EMD Millipore, Darmstadt, Germany). The His-tagged mutant ASC (Y146F) was constructed using a QuikChange Site-Directed Mutagenesis kit (Stratagene) with forward primer 5′- GACGG ATGAG CAGTT CCAGG CAGTG CGGGC -3′ and reverse primer 5′- GCCCG CACTG CCTGG A ACTG CTCAT CCGTC -3′. The mutant sequences are underlined. pENTER-Pyk2-Flag was purchased from ViGene Biosciences. The expression vectors for ASC-Flag and ASC were constructed using the pLKO_AS2 vector (RNAi Core, Taiwan), and that for Flag-NLRP3 was constructed using the pFLAG-CMV2 vector (Sigma).
5
Multiparameter flow cytometry analysis
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Stained cells were analyzed using LSR-Fortessa (BD Bioscience) and analysed with FlowJo software (Tree Star). The following antibodies were used to detect cell surface expression: anti-CD45-PerCPCy5.5, anti-CD11c-PeCy7, anti-Ly6C-FITC, anti-Ly6G-PE and anti-Ly6C-APC (BD Bioscience); anti-Ly6G-APC/Cy7 and anti-CD4-AF700 (Biolegend); anti-CD8-APC, anti-CD11b-PB, anti-IFNγ-PECy7 and c-MET-FITC (Invitrogen); anti-IFNγ-PE, anti-IL-4-FITC (eBioscience) and anti-Annexin V-PeCy7 (eBioscience).
Cell viability was analyzed using the Live/Dead fixable Aqua Dead Cell Stain Kit (Invitrogen) for ex vivo experiments, whereas DAPI (Sigma-Aldrich) was used for in vitro experiments. Parasite-infected cells were identifies based on the DsRed fluorescence of transgenic parasites.
Cell viability was analyzed using the Live/Dead fixable Aqua Dead Cell Stain Kit (Invitrogen) for ex vivo experiments, whereas DAPI (Sigma-Aldrich) was used for in vitro experiments. Parasite-infected cells were identifies based on the DsRed fluorescence of transgenic parasites.
6
Multicolor Flow Cytometry for Apoptosis and Cell Phenotyping
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Double staining of annexin V and PI was performed using Annexin V Apoptosis detection set phycoerythrin (PE)-Cy7 (eBioscience, San Diego, CA, USA) or Brilliant Violet 421™ (BV421) Annexin V (BioLegend, San Diego, CA, USA), and activated caspases detection was performed using a FLICA 660 caspase-1 or caspase-3/-7 assay kit (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's protocol. Stained cells were analyzed with a flow cytometer Cytomics FC 500 (Beckman Coulter, Fullerton, CA, USA). Triple staining of CD11b, Ly6C, and Ly6G for 32D/TetOff-p210 cells was performed using anti-CD11b-BV421, anti-Ly6C-allophycocyanin (APC) (BD Biosciences), and anti-Ly6G-PE (BD Biosciences), respectively. Stained cells were analyzed with a high-speed cell sorter MoFlo AstriosEQ (Beckman Coulter, Fullerton, CA, USA). Triple staining of CD11b, Ly6C, and Ly6G for spleen cells from mice was performed using anti-CD11b-PE (BD Biosciences) anti-Ly6C-APC (BD Biosciences), and anti-Ly6G-fluorescein isothiocyanate (FITC) (BD Biosciences), respectively. Stained cells were analyzed with a flow cytometer Cytomics FC 500 (Beckman Coulter).
7
Comprehensive Cellular Analysis Protocol
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PMA, ATP, 2DG, nigericin, and PhosSTOP were purchased from Sigma/Aldrich Chemical Co. (St. Louis, MO). MSU and alum were purchased from InvivoGen. GSK2837808A, Z-VAD-FMK, and C16 were purchased from MedChemExpress. Anti-CD45-APC and anti-CD11b-BV605 were purchased from BioLegend. Anti-Ly6G-PE was obtained from BD Bioscience.
8
Profiling Myeloid Cells in Murine EAE
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Blood was collected in 2% EDTA tubes from the orbital vein of 16 isoflurane-anesthetized mice at disease onset (clinical score ≥ 0.5) and at the peak of the EAE. After blocking Fc receptors, cells were labeled with the following antibodies: anti-Ly-6C FITC (10 µg/mL, AL-21 clone), anti-Ly-6G PE (4 µg/ml, 1A8 clone), and anti-CD11b PerCP-Cy5.5 (4 µg/ml, M1/70 clone) all from BD Biosciences; anti-MHC-II PE-Cy7 (4 µg/ml, M5/114.15.2 clone), anti-CD11c APC (4 µg/ml, N418 clone), and anti-F4/80 eFluor450 (4 µg/ml, BM8 clone) from eBioscience. Analysis was performed in an FACS Canto II cytometer (BD Biosciences) and data analysis was assessed using FlowJo 10.6.2 software (FlowJo, LLC-BD Biosciences).
9
Flow Cytometry Analysis of Immune Cells
10
Multiparameter Flow Cytometry Protocol
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Anti-CD11b-PacBlu, αCD11b-FITC, αCD4-PE, αCD4-APC, αCD11c-APC, αCD11c-PE, αTCRvα2-PE, and αCD3ε(145-2C11) were purchased from BioLegend (San Diego, CA, USA). Anti-Ly6C-PerCP and αFoxP3-APC were products of eBioscience (San Jose, CA, USA). Anti-Ly6G-PE, αNK1.1-PE, αCD8α-PacBlu, αCD25-PE, and αCD103-Alexa Fluor 647 were products from BD Biosciences (San Jose, CA, USA). Flt3L-Fc fusion protein was purchased from BioXCell (West Lebanon, NH, USA). Anti-IL-2 was purchased from R&D Systems (Minneapolis, MN, USA). Fc-GITR-L fusion protein was produced as described previously (9 (link)).
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