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. 2020 Sep 18;11:578533. doi: 10.3389/fmicb.2020.578533

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Copyright © 2020 Ayoola, Nakamya, Shack, Park, Lim, Lee, Ross, Eoh and Nanduri.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Measurement of surface-exposed phosphocholine (PC) in TIGR4 and deletion strains. Pneumococci were grown to exponential phase and PC was measured by flow cytometry. PC was stained with an unconjugated Kappa murine myeloma IgA anti-phosphocholine antibody, followed by detection with a phycoerythrin (PE)-conjugated rat anti-mouse secondary antibody. Samples were fixed in 2% paraformaldehyde and histogram heights read in blue laser 2 channel (BL2-H) on an Attune Acoustic Focusing Cytometer. The gate was set based on a negative control that was treated with secondary antibody only. Representative histogram overlay plots, from three independent experiments, of the fluorescence intensity of murine myeloma IgA antibody binding to exposed PC on TIGR4 (red), ΔspeE (yellow), and ΔpotABCD (blue) are shown.