cd45r b220 alexa fluor 647  (Bio-Rad)

Journal: Frontiers in Immunology

Article Title: Role of CXCL5 in Regulating Chemotaxis of Innate and Adaptive Leukocytes in Infected Lungs Upon Pulmonary Influenza Infection

doi: 10.3389/fimmu.2021.785457

Figure Lengend Snippet: Pulmonary innate and adaptive immune responses induced by influenza infection in WT and CXCL5 -/- mice. (A) Single lung cells were collected from WT and CXCL5 -/- mice at 3 d.p.i. and analyzed by flow cytometry to detect macrophages (CD64+CD45+), neutrophils (Ly6G+CD45+), NK cells (NK1.1+CD45+), CD4 T cells (CD4+CD45+), CD8 T cells (CD8+CD45+), and CD19 B cells (CD19+CD45+). (B) Numbers of different leukocyte subsets among pulmonary CD45+ leukocytes at 3 d.p.i. (n=4). (C) Microscopic view of BALF leukocytes from WT and CXCL5 -/- mice collected at 3 d.p.i. and cytospun. Black arrows, neutrophils (×400 magnification). (D) Single lung cells were collected from WT and CXCL5 -/- mice at 8 d.p.i. and analyzed by flow cytometry to detect CD4 T cells (CD4+CD45+), CD8 T cells (CD8+CD45+), CD19 B cells (CD19+CD45+), and influenza-specific CD8 T cells (APC-conjugated class I tetramers loaded with the influenza virus NP peptide ASNENMETM). (E) Numbers of different leukocyte subsets among pulmonary CD45+ leukocytes at 8 d.p.i. (n=4). (F) Lung sections from WT and CXCL5 -/- mice were stained at 3 d.p.i. with an anti-CD19 antibody and hematoxylin (×200 magnification). (G) Numbers of NP tetramer-positive CD8+ T cells among pulmonary CD45+ leukocytes at 8 d.p.i. (n=4). (H) The levels of IFN-γ in BALF from WT and CXCL5 -/- mice at 8 d.p.i. were determined by ELISA (n=5). (I) The total numbers of CD4+ T cells and B220+ B cells in the MLNs at 8 d.p.i. were determined by cell counting together with flow cytometry (n=4). (J) The HI titers of the BALF samples from WT and CXCL5 -/- mice at 8 d.p.i. against H1N1 virus were evaluated (n=4). (K) Measurements of influenza-specific total IgG in the BALF of WT and CXCL5 -/- mice infected with the H1N1 virus at 8 d.p.i. (n = 5). (L) The numbers of plasmablasts among pulmonary B cells were detected using anti-CD19 and anti-CD138 antibodies by flow cytometry of samples from WT and CXCL5 -/- mice at 8 d.p.i. (n=4). (M) The total numbers of CD138+CD19+ B cells in the MLNs at 8 d.p.i. were determined by cell counting together with flow cytometry (n=4). The error bars represent the SDs. * P < 0.05 based on Student’s t -test.

Article Snippet: The slides were then incubated with an Alexa Fluor 647-conjugated rat anti-mouse CD45R (B220) monoclonal antibody (557683, BD Biosciences, NJ, USA) and an eFluor 570-conjugated rat anti-mouse CD4 monoclonal antibody (41-9766-82, Thermo Fisher, MA, USA) for 2 h at room temperature.

Techniques: Infection, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Cell Counting

Journal: Frontiers in Immunology

Article Title: Role of CXCL5 in Regulating Chemotaxis of Innate and Adaptive Leukocytes in Infected Lungs Upon Pulmonary Influenza Infection

doi: 10.3389/fimmu.2021.785457

Figure Lengend Snippet: Establishment of adaptive immunity in the infected lungs of WT and CXCL5 -/- mice. (A) Lung lobe sections from WT and CXCL5 -/- mice were stained at 10 d.p.i. with an anti-B220 antibody and hematoxylin (×30 magnification). (B) The positively stained area (iBALT structure) in each lung lobe section was calculated using CaseViewer software (3DHISTECH). The total area of iBALT per lung section was calculated from 4 mice per group (3 lung lobe sections per mouse). (C) Lung sections were stained with anti-B220 (red) and anti-CD4 (yellow) antibodies for immunofluorescence detection. The immunofluorescence sections were counterstained with DAPI (blue). (D) GO functional analysis of upregulated genes in the infected lungs of CXCL5 -/- mice compared to WT mice at 7 and 10 d.p.i. The methods and criteria for filtration of differentially expressed genes are described in the MATERIALS AND METHODS section. (E) Mice were infected with H1N1 or H3N2 (3000 CCID 50 ), and the body weight loss (n=12) of the mice was monitored until 2 weeks post infection. WT-1 H1N1: WT mice infected with H1N1; WT-1 H3N2: WT mice infected with H3N2; CXCL5 -/– 1 H1N1: CXCL5 -/- mice infected with H1N1; CXCL5 -/– 1 H3N2: CXCL5 -/- mice infected with H3N2; WT-2 H1N1: WT mice recovered from H1N1 infection and infected with H1N1; WT-2 H3N2: WT mice recovered from H1N1 infection and infected with H3N2; CXCL5 -/– 2 H1N1: CXCL5 -/- mice recovered from H1N1 infection and infected with H1N1; CXCL5 -/– 2 H3N2: CXCL5 -/- mice recovered from H1N1 infection and infected with H3N2. (F, G) Lung viral loads of H1N1 (F) and H3N2 (G) -infected mice were determined based on the number of influenza M gene RNA copies detected by RT-qPCR at different d.p.i. The error bars represent the SDs of 4 samples. * P < 0.05 based on Student’s t -test.

Article Snippet: The slides were then incubated with an Alexa Fluor 647-conjugated rat anti-mouse CD45R (B220) monoclonal antibody (557683, BD Biosciences, NJ, USA) and an eFluor 570-conjugated rat anti-mouse CD4 monoclonal antibody (41-9766-82, Thermo Fisher, MA, USA) for 2 h at room temperature.

Techniques: Infection, Staining, Software, Immunofluorescence, Functional Assay, Filtration, Quantitative RT-PCR