1/ Using a regular light microscope what is the main difference you expect to ob

Question

1/ Using a regular light microscope what is the main difference you expect to observe when viewing cultures of macrophages infected with amastigotes of either Leishmania donovanior Leishmania mexicana.

2/ If you re-examined these cells using a fluorescent microscope and appropriate fluorochrome-labeled antibodies, identify three markers that characterise the parasite-containing vacuole.

3/ Outline a simple experiment to study the fusogenic potential of the Leishmania containing vacuole with other intracellular compartments.

Explanation / Answer

1/ macrophages with tight vacuoles containing mostly single parasites (L. donovani) vs. large vacuoles containing multiple communal parasites (L. mexicana)

2/ three of: MHCII, LAMP1, cathepsin B/D, vacuolar ATPase, Rab7

3/ culture macrophages; feed with parasites to establish stable infection; feed cells with another endosomal / phagocytic tracer e.g. dextran, latex beads; observe behaviour in real time or after staining using fluorescent microsopy or EM or other approaches of relevance. Quantify data. May also pre-load with the tracer before
adding the parasites.

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