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Week 5

First Week in the Johnston Laboratory

My time in the Johnston laboratory will focus on one particular experiment, the macrophage phagocytosis assay. This week I first shadowed Robbie, the post-doc in Dr. Johnston's lab, and then had a go at doing the assay myself.

Laboratory Work

Macrophage Phagocytosis Assay
This experiment aims to determine if and how wild-type and apm4Δ mutant strains of Candida differ in their interaction with macrophages, a major cellular component of the innate immune system. Macrophages most prominently phagocytose, or "eat", and destroy pathogens in an attempt to protect the body from invading organisms. Due to the fact that: (i) we suspect that the apm4Δ mutant may have a different cell wall composition in comparison to wild-type, some of the components of which I have been examining over the past 4 weeks (e.g. chitin); and (ii) cell wall components are integral for macrophage recognition of Candida; this experiment gives a pathophysiologically relevant context to my findings from the Ayscough laboratory.

Macrophage (red) extending pseudopods towards E.coli (green) in preparation for phagocytosis. Source: Pinterest

 First, I had to prepare the macrophages for the assay, which involved placing glass slides at the base of 30 well plates before adding the cells. The next day I then activated the macrophages with PMA, before doing a 30 minute infection with the Candida. At this stage we used overnight cultures, which means the Candida were in yeast form, and 3 different macrophage:Candida ratios. In order to identify which yeast were on the outside of the macrophages, which would help discriminate between attached and engulfed cells, we stained some of the samples with calcofluor white. I then removed the slides from the base of the wells and mounted them onto glass slides. These have to set overnight before they are ready to visualise the next day. 
Macrophages and Candida Albicans. External Candida are stained with calcofluor white and coloured blue.

 This was very much a trial week as I was familiarising myself with the protocol whilst establishing what conditions e.g. cell ratio to use. Next week I hope to use two stains, to clearly identify engulfed and external Candida, and also have a look at comparing wild-type vs. apm4Δ mutant yeast and hyphae. 

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