cold facs wash buffer  (Thermo Fisher)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glutathione Reductase Promotes Fungal Clearance and Suppresses Inflammation during Systemic Candida albicans Infection in Mice

doi: 10.4049/jimmunol.1701686

Figure Lengend Snippet: A. Phagocytosis of C. albicans by WT and Gsr−/− neutrophils. Purified BM neutrophils were incubated with pHrodo red-tagged C. albicans (MOI=10) at 0°C or 37°C for 30 min, and then labeled with Ly6G–Pacific Blue on ice for 20 min. Cells were subsequently washed twice with cold FACS buffer and analyzed by flow cytometry. Ly6G+ neutrophils cells were analyzed for the presence of pHrodo red-tagged C. albicans. Solid lines depict Gsr+/+ and dashed lines depict Gsr−/− neutrophils. B. Fungal killing assays. Serum-opsonized C. albicans yeast were incubated with either purified BM neutrophils or medium for 60 min at an MOI of 10:1, and then the neutrophils were lyzed by hypo-osmolarity. The mixtures were then serially diluted, and plated onto YPD agar plates to culture at 37°C for approximately 24 h. Viable C. albicans cells were scored by counting the colonies formed on the YPD plates. The percentage of killing was calculated using the medium-treated C. albicans as references. Values are means ± SE. *, p<0.05 (t-test, n=3).

Article Snippet: Subsequently, the cells were washed twice with ice-cold FACS washing buffer prior to flow cytometry on a BD LSR II flow cytometer (BD Biosciences).

Techniques: Purification, Incubation, Labeling, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glutathione Reductase Promotes Fungal Clearance and Suppresses Inflammation during Systemic Candida albicans Infection in Mice

doi: 10.4049/jimmunol.1701686

Figure Lengend Snippet: A. C. albicans-induced cytokine production by WT and Gsr−/− macrophages. WT and Gsr−/− BMDM were stimulated with heat-killed C. albicans (MOI=30, 100) for 24 or 48 h, and cytokines in the medium were quantified by ELISA. *, p<0.05 (t-test, n=4). B. Phagocytosis of C. albicans by WT and Gsr−/− BMDM. WT and Gsr−/− BMDM were incubated with pHrodo red-tagged C. albicans (MOI= 3 or 10) at 37°C for 30 min, and then labeled with Pacific blue-labelled anti-mouse F4/80 on ice for 10 min. Cells were subsequently washed twice with cold FACS buffer and analyzed by flow cytometry. F4/80+ BMDM were analyzed for the presence of pHrodo red-tagged C. albicans. Solid lines depict Gsr+/+ and dashed lines depict Gsr−/− cells.

Article Snippet: Subsequently, the cells were washed twice with ice-cold FACS washing buffer prior to flow cytometry on a BD LSR II flow cytometer (BD Biosciences).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Flow Cytometry