TWiV 470: Just a passing phage

December 3, 2017

Bacteriophage transcytosisThe TWiV ninjas reveal that bacteriophage particles rapidly move across monolayers of eukaryotic cells from different tissues.

Hosts: Vincent Racaniello, Dickson DespommierAlan DoveRich Condit, and Kathy Spindler

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3 comments on “TWiV 470: Just a passing phage

  1. Ben Padman Dec 3, 2017

    Hi there, I heard your questions about the CLEM (Correlative Light Electron Microscopy) experiment from the transcytosis paper and wanted to clear up a few things:

    1) I acquired the optical and electron images a week apart using 2 different microscopes: a Leica SP8 laser scanning confocal and a Hitachi H7500 Transmission Electron Microscope.

    2) I used a modified version of my previously published protocol (DOI: 10.1371/journal.pone.0095967)

    3) Kathy had it right: The sub-cellular locations were relocated after ultrathin sectioning of the resin embedded sample.

    4) When I started the experiment, I was really surprised by how many phages there were inside the cells… yet none of them seemed to correlate directly with the optical data. That’s when we realised that SYBR was pH sensitive. This is a problem, because endosomes are slightly acidic (~pH6 for early endosomes; ~pH5 for late endosomes). The phages were leaking SYBR almost immediately after entering the cells. Hindsight is a cruel mistress; should have used a pH resistant dye.