Journal: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: The frequency of GM-CSF+ Th17 cells is increased in DED mice. Flow cytometry analysis showing GM-CSF+IL-17+CD4+ T cells in the lymph nodes of normal ( white bar ) and DED ( gray bar ) mice (gated on CD4+; n = 6 mice per group; data from one experiment of two is shown).

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Flow Cytometry

Journal: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: Blocking T cell–derived GM-CSF reduces CD11b+ cell maturation, proliferation, and migration in vitro. We isolated CD4+ T cells from the draining lymph nodes of DED mice and stimulated the cells with anti-CD3 antibody overnight. Culture supernatant was then incubated with either anti–GM-CSF or IgG control antibody and then added to a CD11b+ cell culture derived from naïve mice. Flow cytometry analysis shows MHC-II expressing ( A ) CD11b+ cells and ( B ) Ki67+ cells 24 hours after coculture ( gray line : isotype control). In a transwell migration assay, GM-CSF neutralized or control supernatant was cultured with naïve CD11b+ cells ( upper well ). One hour later, CD11b+ cells that migrated to the lower well were enumerated. (Each group run in triplicate, data from one experiment of two is shown). CON, IgG isotype control; α-GM-CSF, anti–GM-CSF.

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Blocking Assay, Derivative Assay, Migration, In Vitro, Isolation, Incubation, Cell Culture, Flow Cytometry, Expressing, Transwell Migration Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: Topical neutralization of GM-CSF in a murine model of DED decreases CD11b+ cell maturation and migration to the ocular surface. We induced DED for 7 days and mice were treated topically 3 times daily with anti–GM-CSF or isotype control. Flow cytometry was used to determine frequencies and expression of MHC-II by ( A ) corneal CD11b+ cells and ( B ) frequencies of conjunctival CD11b+ cells. Gray line : isotype. n = 5 mice per group; data from one of two experiments is shown. CON, IgG isotype control; α-GM-CSF, anti–GM-CSF.

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Neutralization, Migration, Flow Cytometry, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: Topical treatment with anti–GM-CSF reduces pathogenic Th17 cells and improves clinical signs of DED. We induced DED for 7 days and mice were treated topically 3 times daily with anti–GM-CSF or isotype control. ( A ) Flow cytometry was used to determine frequencies of IL-17+ and CD4+ Th17 cells in the draining lymph nodes of treated versus control mice. ( B ) Clinical severity of DED after treatment with IgG isotype control ( gray ) versus anti–GM-CSF ( white ) was evaluated using CFS and the NEI scoring rubric (based on a scale of 0–15). Anti–GM-CSF significantly decreases corneal epitheliopathy compared to IgG control ( n = 10 eyes per group; data from one of two experiments is shown). CON, IgG isotype control; α-GM-CSF, anti–GM-CSF.

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Flow Cytometry