Skip to main content

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. 

Comments

Post a Comment

Popular posts from this blog

Introduction to the Project: The Role of Endocytosis in Regulating Cell Wall Composition in Candida Albicans

Background Candida Albicans (C. albicans) C. albicans is the most common human fungal pathogen, which causes various types of Candidiasis depending on the anatomical site of disease manifestation. It is perhaps most readily associated with thrush, which occurs in the throat and vagina. Both in vitro  and in vivo, Candida are capable of growing in a variety of morphogenic forms, namely yeast, pseudohyphae and true hyphae. These forms can be selectively induced by particular experimental conditions; for example, growing C. albicans at 28 o C and 37 o C induces yeast and true hyphal forms, respectively. Pseudohyphal, Yeast, and True Hyphal Forms of Candida albicans . Source: Sudbery, 2011   Endocytosis, AP-2 and Polarised Growth Yeast budding and hyphal growth are highly polarised events, that are co-ordinated in-part by the regulation of cell surface composition via endocytosis (membrane internalisation) and exocytosis (secretion). Both processes are highly complex
Post a Comment
Read more

Week 3

Laboratory Work Experiment 1: Caspofungin Growth Curve The caspofungin plate spotting assay we set up last week did not show any differences between wild-type and apm4 Δ  mutant strains in their ability to grow in the presence of various concentrations of caspofungin. Wild-type and apm4Δ Strains Appear to Grow Similarly  in the Presence of Caspofungin. Wild-type (1st and 3rd rows) and apm4Δ mutant (2nd and 4th rows) strains were grown at 37˚C on a YPD plate supplemented with 0.16 µg/mL caspofungin. Because of this, we decided to do a literature search to find other caspofungin sensitivity assays in order to double check our results. With help from Harriet, I adapted an experiment which measured the OD 600  of cells growing in YPD media supplemented with various concentrations of caspofungin. As it would not be accurate to measure the OD of hyphal cells as an indication of total population size, we could only do this experiment with yeast. In addition, due to the fact that
Post a Comment
Read more

Week 2

BMS Away Day Firth Court Entrance At the beginning of the week I attended the Biomedical Science (BMS) research away day, which was held in Firth Hall. The day consisted of numerous talks and poster presentations given by various members of the four research areas which exist within the BMS department at the University of Sheffield. Namely, cell biology and cancer, developmental biology and disease, neuroscience, and stem cell and regenerative medicine. The day was really interesting as it allowed me to explore the work that is going on within the department; in addition to giving me an indication as to how to approach poster and flash talk presentations when I (hopefully) become a PhD student.   Laboratory Work Experiment 1: Microscopy This week I had my first go at microscopy; using standard transmitted light before moving on to stain some samples and use fluorescence. However, I wasn't able to gain any results from these latter experiments as our cells were co
Post a Comment
Read more