Quantifying Capsule Shedding in Cryptococcus

The relative amount of capsule shedding in the cell supernatant was assayed as previously described [21 (link),30 (link)]. Briefly, capsule induced cultures (incubated as described above) were incubated at 70°C for 15 minutes to denature enzymes, after which the cells were pelleted and the supernatant was sterile filtered. This conditioned medium was then run on a 0.6% agarose gel for 15 hours at 25 V, followed by transfer to a positively charged nylon membrane using Southern blotting methods. The membrane was air dried overnight, followed by blocking with 5% skim milk in Tris-Buffered Saline-Tween-20 (TBST). To detect capsule polysaccharide, blots were incubated with a mouse monoclonal anti-GXM antibody, MAb18B7 (1 μg/ml) [92 (link),93 (link)] for 1 hour, washed 3x with TBST, and incubated with an anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) for 1 hour. Blots were washed 3x with TBST and capsule polysaccharide was detected by enhanced chemiluminescence (ECL Prime Western blotting detection reagent; GE Healthcare).