cd45 pe  (Miltenyi Biotec)

Journal: Cells

Article Title: Expression of Human CEACAM Receptors Promotes Inflammation and Organ Damage During Systemic Candida albicans Infection in Mice

doi: 10.3390/cells15080707

Figure Lengend Snippet: CEABAC10 mice show enhanced kidney inflammation during systemic C. albicans infection. CEABAC10 mice and wild-type littermates were either injected with PBS or infected with 1 × 10 4 CFU/g body weight and were sacrificed after 24 h or 72 h. ( A – D ) Kidney sections were hematoxylin–eosin-stained (representative sections shown in ) and analyzed. ( A – D ). Sections were scored for the degree of renal inflammation ( A ) and analyzed for inflammatory foci ( B , C ) and total area of inflammation ( D ). ( E – G , I ) Concentrations of IL-6 ( E ), IL-1β ( F ) and CCL2/MCP-1 ( G ) and CFUs ( I ) were determined in kidney homogenates by multiplex assay (Luminex)/ELISA and serial plating (additional cytokines shown in ). Note that non-infected kidneys did not show fungal growth ( I ). ( H ) % Ly6G + neutrophils of CD45 + leukocytes isolated from kidneys analyzed by flow cytometry (additional immune cell populations shown in , gating in ). ( J , K ) Representative Grocott silver-stained sections 72 h p.i and blow-ups of the indicated regions. ( L ) Grocott silver-stained sections were scored for the occurrence of hyphal growth. Note that non-infected kidneys did not show fungal growth. ( M – O ) Immunohistochemical staining of consecutive sections of CEABAC10 kidneys 72 h p.i for CEACAM6 ( M , N ) and neutrophil elastase ( O ). Panels display representative images (N = 3). Note that only viable neutrophils are NE + , but that CEACAM6 + cells include viable and dead neutrophils, monocytes, and macrophages and that CEACAM6 + cells ( N ) outnumber viable neutrophils ( O ) by ca. one or two orders of magnitude. Statistics: ( A , K ) Kruskal–Wallis and Dunn’s Multiple Comparison Test, # p < 0.05, ## p < 0.01; ( B – H , L ) One-Way ANOVA and Bonferroni’s Multiple Comparison Test: ** p < 0.01, *** p < 0.005, **** p < 0.001. ( A – G ) Data points with means and standard deviations. ( I , L ) Data points with medians and means. Data are combined from two independent experiments.

Article Snippet: Cells were stained and analyzed on an Attune Acoustic Focusing Cytometer (Life Technologies, Thermo Fisher Scientific, Darmstadt, Germany) using the Attune software v2.1, as described in the respective figure legends with the following antibodies or the corresponding isotype controls: CD45-PE, Ly-6G-PerCP-Vio700, CD3ε-PE-Vio770, CD19-APC, F4/80-FITC, CD45-PE, CD11c-PE-Vio770, CD335-APC (all REA, Miltenyi Biotec, Bergisch Gladbach, Germany), and viability dye eFluor780 (eBioscience/Thermo Fischer GmbH, Darmstadt, Germany).

Techniques: Infection, Injection, Staining, Multiplex Assay, Luminex, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Immunohistochemical staining, Comparison

Journal: Cells

Article Title: Expression of Human CEACAM Receptors Promotes Inflammation and Organ Damage During Systemic Candida albicans Infection in Mice

doi: 10.3390/cells15080707

Figure Lengend Snippet: CEABAC10 livers display enhanced inflammation, acute coagulation necroses with immune cell infiltration, and multifocal hemorrhage during systemic C. albicans infection. CEABAC10 mice and wild-type littermates were either injected with PBS or infected with 1 × 10 4 CFU/g body weight and were sacrificed after 24 h or 72 h. ( A – C ) Occurrence of macroscopic pathologic liver abnormalities (white areas) observed during necropsy 72 h p.i. (18 CEA, 14 WT). ( A , B ) Representative images. ( C ) Livers without macroscopic abnormalities (open bars) and livers displaying white areas 72 h p.i. (filled bars). ( D – H ) Liver sections were hematoxylin–eosin-stained and analyzed. ( D – F ) Representative CEABAC10 liver section 72 h p.i. showing acute coagulation necroses with immune cell infiltration and multifocal hemorrhage. ( G , H ) Sections were scored for the degree of acute coagulation necroses ( G ) and for the grade of acute purulent necrotizing hepatitis ( H ). ( I , J ) % Ly6G + neutrophils ( I ) and F4/80 + macrophages ( J ) of CD45 + leukocytes isolated from livers (additional immune cell populations shown in , gating in ). ( K – M ) Concentrations of IL-6 ( K ), CCL2/MCP-1 ( L ) and CCL3/MIP-1alpha ( M ) were determined in liver homogenates (additional cytokines shown in ). ( N ) CFUs in liver homogenates. ( O – Q ) Immunohistochemical staining of consecutive CEABAC10 liver sections 72 h p.i. for CEACAM6 ( O , P ) or neutrophil elastase (NE) ( Q ) (representative sections, N = 3). Note that only viable neutrophils are NE + , but that CEACAM6 + cells include viable and dead neutrophils and macrophages/monocytes and that CEACAM6 + cells ( P ) outnumber viable neutrophils ( Q ) by one or two orders of magnitude. ( R ) Spleen sections were hematoxylin–eosin-stained and scored for splenitis. ( S ) CFUs were determined in spleen homogenates. Note that livers and spleens from uninfected animals (8 WT and 8 CEABAC10 for PBS) did not display any fungal growth and that none of the infected liver and spleen samples displayed any hyphal growth. Data are combined from two ( G – N , R , S ) or four ( C ) independent experiments. Statistics: ( C ) Fisher’s Exact Test, two-sided $$$$ p < 0.001; ( G , H , R ) Kruskal–Wallis and Dunn’s Multiple Comparison Test: # p < 0.05, ## p < 0.01; ( I – N , S ) One-Way ANOVA and Bonferroni’s Multiple Comparison Test: * p < 0.05, ** p < 0.01, *** p < 0.005. For ( L , S ), log data were used for statistical analysis. ( G – N , R , S ) Data points with means and standard deviations.

Article Snippet: Cells were stained and analyzed on an Attune Acoustic Focusing Cytometer (Life Technologies, Thermo Fisher Scientific, Darmstadt, Germany) using the Attune software v2.1, as described in the respective figure legends with the following antibodies or the corresponding isotype controls: CD45-PE, Ly-6G-PerCP-Vio700, CD3ε-PE-Vio770, CD19-APC, F4/80-FITC, CD45-PE, CD11c-PE-Vio770, CD335-APC (all REA, Miltenyi Biotec, Bergisch Gladbach, Germany), and viability dye eFluor780 (eBioscience/Thermo Fischer GmbH, Darmstadt, Germany).

Techniques: Coagulation, Infection, Injection, Staining, Isolation, Immunohistochemical staining, Comparison

Journal: Cells

Article Title: Expression of Human CEACAM Receptors Promotes Inflammation and Organ Damage During Systemic Candida albicans Infection in Mice

doi: 10.3390/cells15080707

Figure Lengend Snippet: Increased numbers of CEACAM6 + myeloid cells in organs of CEABAC10 mice during systemic C. albicans infection and loss of CEACAM6 + liver macrophages. CEABAC10 mice were either injected with PBS or infected with 1 × 10 4 CFU/g body weight and were sacrificed after 24 h or 72 h. Immune cells were isolated from kidneys ( A – C ), liver ( D – F ), and spleen ( G – I ) and stained with viability dye/CD45/CD11b/Ly6G and viability dye/CD45/F4/80/Ly6C/CD11c. Neutrophils ( A , D , G ), monocytes ( B , E , H ), and macrophages ( C , F , I ) were analyzed for their percentage of human CEACAM6-positive cells (gating: see ). Graphs show the percentages of CEACAM6-positive cells for the respective cell types with means and standard deviations. Data are from one experiment. Statistics: One-Way ANOVA and Bonferroni’s Multiple Comparison Test: * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

Article Snippet: Cells were stained and analyzed on an Attune Acoustic Focusing Cytometer (Life Technologies, Thermo Fisher Scientific, Darmstadt, Germany) using the Attune software v2.1, as described in the respective figure legends with the following antibodies or the corresponding isotype controls: CD45-PE, Ly-6G-PerCP-Vio700, CD3ε-PE-Vio770, CD19-APC, F4/80-FITC, CD45-PE, CD11c-PE-Vio770, CD335-APC (all REA, Miltenyi Biotec, Bergisch Gladbach, Germany), and viability dye eFluor780 (eBioscience/Thermo Fischer GmbH, Darmstadt, Germany).

Techniques: Infection, Injection, Isolation, Staining, Comparison