Journal: Journal of dairy science

Article Title: Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity.

doi: 10.3168/jds.2022-22728

Figure Lengend Snippet: Figure 1. Blood neutrophils expressing major histocompatibility complex class II (MHC-IIpos PMN) display better phagocytosis activity than MHC-IIneg (i.e., not expressing MHC-II) PMN in Holstein and Normande cows. Cells were prepared from blood from the 2 breeds. (A) Proportion of total PMN in blood was determined by complete blood count, and (B) percentage of MHC-IIpos PMN was determined by flow cytometry among CD45+ G1+ live cells. (C, D) Phagocytosis of pHrodo Escherichia coli BioParticles by total PMN (C) or MHC-IIpos and MHC-IIneg PMN was assessed by measuring fluorescence intensity directly correlated with ingested particles. (A–D) Data are medians with interquartile range (n = 5 Holstein, n = 7 Normande); data are representative of 1 experiment over 2 independent experiments. **P < 0.01 (Wilcoxon signed-rank test). MFI = mean fluorescence intensity.

Article Snippet: Antibodies used for flow cytometry analysis Antibody Target1 Reactivity Clone Supplier Isotype Dye2 Primary antibodies Granulocytes (G1) Bovine CH138A KingFisher Biotech Mouse IgM — MHC-II Feline CAT82A KingFisher Biotech Mouse IgG1 — CD14 Human CC-G33 Bio-Rad Mouse IgG1 PE–Alexa Fluor 750 CD45 Sheep 1.11.32 Bio-Rad Mouse IgG1 PE CD3 Bovine MM1A KingFisher Biotech Mouse IgG1 — Secondary antibodies IgM Mouse — Invitrogen Goat IgG Alexa Fluor 647 IgG1 Mouse — Invitrogen Goat IgG Alexa Fluor 488 IgG1 Mouse — Invitrogen Goat IgG PE Fixable viability dye (FVD) — — BioLegend — Zombie aqua FVD — — eBiosciences — efluor 780 1CD14 and CD45 antibodies are directly conjugated, whereas secondary antibodies were used to measure G1, MHC-II, and CD3 expression.

Techniques: Expressing, Immunopeptidomics, Activity Assay, Flow Cytometry, Fluorescence

Journal: Journal of dairy science

Article Title: Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity.

doi: 10.3168/jds.2022-22728

Figure Lengend Snippet: Figure 2. Neutrophils expressing major histocompatibility complex class II (MHC-IIpos PMN) have higher bactericidal capacity than MHC- IIneg (i.e., not expressing MHC-II) PMN. (A) Flow cytometry gating strategy used to identify MHC-IIneg and MHC-IIpos PMN in milk. (B) After labeling milk cells, phagocytosis by MHC-IIpos or MHC-IIneg PMN was assessed using the Escherichia coli P4-GFP (green fluorescent protein) strain at 37°C. Phagocytosis by PMN was normalized on PMN stimulated with E. coli P4-GFP at 4°C. Data represent n = 2 experiments with n = 7 cows. (C) Oxidative stress was measured in MHC-IIpos and MHC-IIneg neutrophils using the CellROX Orange probe, which reacts with all reactive oxygen species (ROS). Cells were activated with tert-butyl hydroperoxide (TBHP), and levels of ROS were measured by flow cytom- etry. Data represent n = 2 experiments with n = 8 cows. (B, C) Fluorescence for each sample is depicted, and paired MHC-IIpos and MHC-IIneg samples were analyzed for each animal. Data are medians with interquartile range. FSC-A = forward scatter, area; FSC-H = forward scatter, height; SSC-A = side scatter, area; FVD = fixable viability dye. Dots = MHC-IIneg PMN; squares = MHC-IIpos PMN. *P < 0.05; **P < 0.01 (Wilcoxon signed-rank test).

Article Snippet: Antibodies used for flow cytometry analysis Antibody Target1 Reactivity Clone Supplier Isotype Dye2 Primary antibodies Granulocytes (G1) Bovine CH138A KingFisher Biotech Mouse IgM — MHC-II Feline CAT82A KingFisher Biotech Mouse IgG1 — CD14 Human CC-G33 Bio-Rad Mouse IgG1 PE–Alexa Fluor 750 CD45 Sheep 1.11.32 Bio-Rad Mouse IgG1 PE CD3 Bovine MM1A KingFisher Biotech Mouse IgG1 — Secondary antibodies IgM Mouse — Invitrogen Goat IgG Alexa Fluor 647 IgG1 Mouse — Invitrogen Goat IgG Alexa Fluor 488 IgG1 Mouse — Invitrogen Goat IgG PE Fixable viability dye (FVD) — — BioLegend — Zombie aqua FVD — — eBiosciences — efluor 780 1CD14 and CD45 antibodies are directly conjugated, whereas secondary antibodies were used to measure G1, MHC-II, and CD3 expression.

Techniques: Expressing, Immunopeptidomics, Flow Cytometry, Labeling, Fluorescence

Journal: Journal of dairy science

Article Title: Neutrophils expressing major histocompatibility complex class II molecules circulate in blood and milk during mastitis and show high microbicidal activity.

doi: 10.3168/jds.2022-22728

Figure Lengend Snippet: Figure 3. Neutrophils expressing major histocompatibility complex class II (MHC-IIpos PMN) are present in blood and milk during clinical mastitis and their proportion does not vary over time. (A) Description of sampling protocol. Blood and milk samples from the infected quarter (S1) were immediately collected after the diagnosis of the clinical mastitis. After antibiotic treatment for 5 milkings of the infected quarter, second blood and milk samples (S2) were collected between d 3 and 6 (the day was chosen to avoid weekends), and third blood and milk samples (S3) were collected at d 21. (B) Flow cytometry gating strategy used to identify different cells populations in blood and in milk. Blood MHC- IIpos PMN are defined as CD45+ SCChigh FSChigh G1+ MHC-II+. Eosinophils were excluded by excitation with violet laser in the “granulocytes” gate. Milk PMN, macrophages, and T lymphocytes are defined as CD45+ G1+ CD14−, CD45+ G1− CD14+, and CD45+ CD3+, respectively. Milk MHC-IIpos PMN are defined as MHC-II+ in the “PMN” gate. (C) Percentage of PMN, macrophages, and T lymphocytes in milk at each sample collection time. Red dots = milks with SCC >300,000 cells/mL; blue dots = milks with SCC <300,000 cells/mL. (D) Percentage of MHC-IIpos PMN in blood and milk at each time point. (C, D) Data are boxes, and whiskers show minimum to maximum range of values (n = 10 cows and 27 independent experiments). FSC-A = forward scatter, area; FSC-H = forward scatter, height; SSC-A = side scatter, area; FVD = fixable viability dye.

Article Snippet: Antibodies used for flow cytometry analysis Antibody Target1 Reactivity Clone Supplier Isotype Dye2 Primary antibodies Granulocytes (G1) Bovine CH138A KingFisher Biotech Mouse IgM — MHC-II Feline CAT82A KingFisher Biotech Mouse IgG1 — CD14 Human CC-G33 Bio-Rad Mouse IgG1 PE–Alexa Fluor 750 CD45 Sheep 1.11.32 Bio-Rad Mouse IgG1 PE CD3 Bovine MM1A KingFisher Biotech Mouse IgG1 — Secondary antibodies IgM Mouse — Invitrogen Goat IgG Alexa Fluor 647 IgG1 Mouse — Invitrogen Goat IgG Alexa Fluor 488 IgG1 Mouse — Invitrogen Goat IgG PE Fixable viability dye (FVD) — — BioLegend — Zombie aqua FVD — — eBiosciences — efluor 780 1CD14 and CD45 antibodies are directly conjugated, whereas secondary antibodies were used to measure G1, MHC-II, and CD3 expression.

Techniques: Expressing, Immunopeptidomics, Sampling, Infection, Biomarker Discovery, Flow Cytometry