Journal:

Article Title: Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival ▿ †

doi: 10.1128/IAI.00088-09

Figure Lengend Snippet: SP-D opsonization decreases cap59Δ cell colocalization with LAMP-1 and decreases fungal breakdown. Lysosomal trafficking of SP-D-bound or unbound AF647-labeled cap59Δ cells was assessed by immunofluorescence and confocal microscopy analyses. The percent colocalization and percent debris were determined using the colocalization and regions tools, respectively, in Metamorph software. The percent debris was obtained by creating regions around intact cap59Δ cells in the red channel and subtracting the resultant value from the number of all red pixels present for a given image to obtain the pixel value for debris. This value was then divided by the total number of red pixels and multiplied by 100 to obtain a percentage. Results were obtained by assessing 15 randomly selected cells under each condition and are presented as the means ± standard errors. Similar results were obtained from two experiments performed in triplicate. Data were analyzed using the two-tailed Student t test, and a P value of <0.05 was considered significant. Scale bars, 5 μm. DIC, differential interference contrast image; Cn, C. neoformans; +, with; −, without.

Article Snippet: Next, the macrophages were incubated with monoclonal rat anti-mouse CD107a (LAMP-1) primary antibody (Pharmingen) at a final concentration of 1 μg/ml for 30 min at 37°C, washed three times with blocking solution, and incubated with AF488-labeled goat anti-rat secondary antibody (Invitrogen), diluted 1:2,500, for 30 min at 37°C.

Techniques: Labeling, Immunofluorescence, Confocal Microscopy, Software, Two Tailed Test