An aliquot of each whole blood sample (30 μl) was incubated for 15 min on ice with the following antibodies (BioLegend, San Diego, CA, USA): PE-labelled anti-mouse CD45 (clone 30-F11), PE/Cy7 anti-mouse/human CD11b (clone M1/70), APC anti-mouse Ly-6G (clone 1A8) and APC/Fire750 anti-mouse Ly-6C (clone HK1.4). Negative control staining was performed using appropriate isotype antibodies (BioLegend). The stained blood samples were lysed with a red blood cells lysis buffer (Tonbo Biosciences) and analyzed using a Gallios flow cytometer and Kaluza software (Beckman Coulter, Inc., Brea, CA, USA). Cells were defined with the following stains [25 (link)]: the gated CD45+CD11b+ cells as total white blood cells; CD45+CD11b+Ly-6G+ as neutrophils; CD45+CD11b+Ly-6G-Ly-6C+ as monocytes. The gating strategy for these neutrophils and monocytes was summarized in S2 Fig .
Pe cy7 anti mouse human cd11b clone m1 70
Manufactured by BioLegend
Sourced in United States
PE/Cy7 anti-mouse/human CD11b (clone M1/70) is a fluorochrome-conjugated antibody that binds to the CD11b cell surface antigen, which is expressed on myeloid cells such as monocytes, macrophages, and neutrophils. This antibody can be used for the identification and enumeration of CD11b-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza software, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Apc anti mouse ly6g clone 1a8, by BioLegend (1 mentions)
Anti cd16 cd32 antibody, by BioLegend (1 mentions)
Pe anti mouse f4 80 clone bm8, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Liberase, by Roche (1 mentions)
Zombie yellow fixable viability kit, by BioLegend (1 mentions)
Anti mouse cd16 cd32 clone 93, by BioLegend (1 mentions)
3 protocols using pe cy7 anti mouse human cd11b clone m1 70
1
Flow Cytometric Analysis of Immune Cells
2
Liver Leukocyte Immunophenotyping Protocol
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The leukocyte single cell suspensions from the livers were obtained, as described elsewhere [36 (link)]. After treatment with anti-CD16/CD32 antibodies (1/100, clone 93, Biolegend), the cells were stained for 30 min at 4 °C with combinations of the following antibodies (1/100): eFluor 660 anti-mouse CD8a (clone 53-6.7); eFluor 660 anti-mouse F4/80 (clone BM8) from ThermoFisher Scientific; Brilliant Violet 421 anti-mouse NK-1.1 (clone PK136); Alexa Fluor 488 anti-mouse Ly6C (clone HK1.4); PE anti-mouse F4/80 (clone BM8); PerCP anti-mouse Ly6G (clone 1A8); PE/Cy7 anti-mouse/human CD11b (clone M1/70); APC/Cy7 anti-mouse CD45 (clone 30-F11); and isotype controls from Biolegend. The data analysis was performed in Summit 5.2.0 (Beckman-Coulter, Fullerton, CA, USA). The distinct cells’ populations were gated, as shown in Figure S3A, Supplementary Materials .
3
Isolation and Characterization of Ear Immune Cells
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Untreated ears or ears that were intradermally injected with fluorescently labeled αOVA-mIgE 16 h prior were separated into dorsal and ventral halves and minced before digestion with Liberase (Roche) and subjected to disruption, to generate single cell suspensions, as previously described (Riol-Blanco et al., 2014 (link)). Suspension cells were resuspended in PBS and incubated with Zombie Yellow Fixable Viability kit (BioLegend) before incubation with anti–mouse CD16/CD32 (clone 93) in FACS buffer (2 mM EDTA and 0.5% BSA in PBS). Antibodies for surface antigen staining included Alexa Fluor 647 anti–mouse CD117 (c-Kit; clone 2B8; BioLegend), Pacific Blue anti–mouse CD45 (clone 30-F11; BioLegend), PE/Cy7 anti–mouse/human CD11b (clone M1/70; BioLegend), FITC anti–mouse CD8 (clone 53-6.7; BioLegend), PE anti–mouse CD8 (clone 53-6.7; BioLegend), APC anti–mouse CD8 (clone 53-6.7; BioLegend), and PE/Cy7 anti–mouse CD8 (clone 53-6.7; BioLegend). Cells were resuspended in FACS buffer after staining and acquired using an LSRII flow cytometer (BD). Data were analyzed using FlowJo version 7.6 software (Tree Star).
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