Final Weeks
I decided to upload just one post covering the final 3 weeks, as they were very similar in that I spent them repeating the macrophage phagocytosis assay and analysing the images I obtained from microscopy.
Laboratory Work
Macrophage Phagocytosis - Calcofluor White and Concavalin A + FITC Staining
This protocol was very similar as I described for week 5, except it involved additional cell permeabilization and second staining steps. Following infection with Candida yeast, samples were stained with calcofluor white, meaning that all external Candida were coloured blue. Then, I fixed the samples and permeabilized the cells before applying the second stain, which was concavalin A conjugated to FITC, which coloured both internal and external Candida green. As concavalin A binds to residues present in the macrophage as well as the Candida, these also became visible with the conA + FITC staining.
Although in the images above, the calcoflour white staining was very bright and distinguishable, this was not always the case, and so I changed the colour of the calcoflour white to red, and the conA + FITC to yellow, so I could more easily distinguish where the two channels overlapped.
This allowed me to easily quantify: (i) contact number; (ii) infection burden; and (iii) the % of macrophages infected. Although I did also do this experiment with hyphae several times, I found the analysis stage a lot more difficult, and the images did not come out as well in comparison to the yeast. Of all the analysis I did, I found there to be no consistently significant difference in infection burden or contact number when macrophages were infected with apm4Δ/Δ vs wild-type yeast. However, I did find that a significantly smaller % of macrophages were infected with apm4Δ/Δ vs wild-type Candida.
Final Thoughts on Summer Placement
This studentship allowed me to spend my summer indulging my interests, whilst gaining invaluable skills in areas I previously had little to no experience in. I now feel more confident and equipped to pursue PhD study and ultimately a career in academia. I am incredibly grateful to Prof. Ayscough, Dr. Johnston, Ms. Knafler and Dr. Evans for their supervision and guidance throughout this project; and of course to the Biochemical Society for the financial support provided by the studentship grant. If anyone reading this ever finds themselves in a position where they are considering undertaking a summer placement, I really cannot recommend it enough.
I decided to upload just one post covering the final 3 weeks, as they were very similar in that I spent them repeating the macrophage phagocytosis assay and analysing the images I obtained from microscopy.
Laboratory Work
Macrophage Phagocytosis - Calcofluor White and Concavalin A + FITC Staining
This protocol was very similar as I described for week 5, except it involved additional cell permeabilization and second staining steps. Following infection with Candida yeast, samples were stained with calcofluor white, meaning that all external Candida were coloured blue. Then, I fixed the samples and permeabilized the cells before applying the second stain, which was concavalin A conjugated to FITC, which coloured both internal and external Candida green. As concavalin A binds to residues present in the macrophage as well as the Candida, these also became visible with the conA + FITC staining.
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| External Candida Stained with Calcofluor White |
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| External and Internal Candida and Macrophages Stained with ConA + FITC |
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| Overlay of the two Images, External Candida in Blue and Internal Candida and Macrophages in Green |
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| Close up of Overlay Images |
Although in the images above, the calcoflour white staining was very bright and distinguishable, this was not always the case, and so I changed the colour of the calcoflour white to red, and the conA + FITC to yellow, so I could more easily distinguish where the two channels overlapped.
 |
| Candida stained with calcoflour white and con A + FITC, corresponding to red and yellow, respectively. |
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| Close up of Above Image |
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| APM4 Contributes to the Phagocytosis of C. albicans by Macrophages. Macrophages were infected for 30 minutes with yeast cells and then stained with calcoflour white and concavalin A + FITC to detect external and engulfed Candida, respectively. Values plotted are the mean and standard deviation obtained from 4 and 5 independent experiments with wild-type and apm4Δ/Δ strains, respectively. * p = 0.0263 (Unpaired t test). |
Final Thoughts on Summer Placement
This studentship allowed me to spend my summer indulging my interests, whilst gaining invaluable skills in areas I previously had little to no experience in. I now feel more confident and equipped to pursue PhD study and ultimately a career in academia. I am incredibly grateful to Prof. Ayscough, Dr. Johnston, Ms. Knafler and Dr. Evans for their supervision and guidance throughout this project; and of course to the Biochemical Society for the financial support provided by the studentship grant. If anyone reading this ever finds themselves in a position where they are considering undertaking a summer placement, I really cannot recommend it enough.
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