Nine to ten week old male C57BL/6 and Nlrp3−/− mice were injected i.p. with 500 μM Gd-DTPA in phosphate buffered saline (PBS). PBS injected C57BL/6 served as controls. After 24 hours, mice were euthanized and cells were collected from the peritoneal cavity. Cells were stained for flow cytometry with anti-CD11b, anti-Ly6G (BD Biosciences, San Jose, CA, USA), and anti-Ly6C (AbDSerotec, Raleigh, NC, USA), recorded on a LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA).
Anti ly6c
Manufactured by Bio-Rad
Sourced in United States, Spain
Anti-Ly6C is a laboratory reagent used for the detection and analysis of Ly6C, a protein marker expressed on various cell types. It can be used in flow cytometry and other immunological applications to identify and characterize Ly6C-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 10, by BioLegend (2 mentions)
Ionomycin phorbol 12 myristate 13 acetate brefeldin a, by BioLegend (2 mentions)
Foxp3 fix perm, by BioLegend (2 mentions)
Rbc lysis, by Thermo Fisher Scientific (2 mentions)
Anti il 17, by BioLegend (2 mentions)
Anti cd4, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by BioLegend (2 mentions)
Anti cd11b, by BioLegend (2 mentions)
Anti ly6g, by BioLegend (2 mentions)
Anti cd8, by BioLegend (2 mentions)
Anti cd44, by BioLegend (2 mentions)
Anti cd62l, by BioLegend (2 mentions)
Anti ly6g, by BD (1 mentions)
Anti cd11b, by BD (1 mentions)
Anti foxp3, by BioLegend (1 mentions)
Anti cd115, by Thermo Fisher Scientific (1 mentions)
A2547, by Merck Group (1 mentions)
Anti α sma, by Merck Group (1 mentions)
4 protocols using anti ly6c
1
Gd-DTPA-Induced Peritoneal Leukocyte Analysis
2
Multiparametric Flow Cytometry of Immune Cells
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Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5
min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS
buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer
were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with:
anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all
BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend;
20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then
anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated
±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h,
37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8
(20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and
anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or
Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with
anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend),
anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing,
and analysis by flow cytometry (Accuri C6).
min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS
buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer
were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with:
anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all
BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend;
20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then
anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated
±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h,
37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8
(20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and
anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or
Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with
anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend),
anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing,
and analysis by flow cytometry (Accuri C6).
3
Immunophenotyping Murine Splenocytes
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Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5 min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS buffer (1% BSA, 0.05% NaN3, in PBS) or full RPMI 1640. Cells in FACS buffer were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with: anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend; 20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated ±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h, 37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8 (20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or Ly6C+ cells, whole blood (ethylenediaminetetraacetic acid) was stained with anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend), anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing, and analysis by flow cytometry (Accuri C6).
4
Histological Evaluation of Liver Fibrosis
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Hepatic histology was evaluated on 4-μm sections of 4% p-formaldehyde-fixed liver which were blocked in paraffin wax and stained with Hematoxylin and Eosin (H&E), Masson's Trichrome or Sirius Red. The stage of fibrosis was scored in a blinded manner by an experienced pathologist using a four-point severity scale (0, normal; 1, mild; 2, moderate; 3, severe) for necrosis, inflammatory cell infiltration, bile duct hyperplasia and fibrosis [34 (link)]. Bile infarcts were quantified in H&E-stained sections and the percentage of the focal necrosis surface to the whole liver section area was assessed. Liver immunohistochemistry was performed as previously described [26 (link)]. Random fields (4 per mice) were chosen for quantification of immunostained positive cells. The antibodies used were anti-F4/80 (MCA497, Serotec), anti-Ly6C (provided by A. Castrillo, CSIC, Spain) and anti-α-SMA (A2547, Sigma).
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