PMID- 33441638
OWN - NLM
STAT- MEDLINE
DCOM- 20210816
LR  - 20210816
IS  - 2045-2322 (Electronic)
IS  - 2045-2322 (Linking)
VI  - 11
IP  - 1
DP  - 2021 Jan 13
TI  - Tailored co-localization analysis of intracellular microbes and 
      punctum-distributed phagosome-lysosome pathway proteins using ImageJ plugin 
      EzColocalization.
PG  - 1096
LID - 10.1038/s41598-020-79425-5 [doi]
LID - 1096
AB  - Immunofluorescence is indispensable to monitor redistribution of proteins 
      involved in phagosome-lysosome association pathway-relevant (P-LApr) proteins. 
      The software digitizing the signals of these proteins in an unbiased and 
      automated manner is generally costly and not widely available. The open-source 
      ImageJ plugin EzColocalization, which is for co-localization analysis of 
      reporters in cells, was not straightforward and sufficient for such analysis. We 
      describe here the input of custom Java code in a novel tailored protocol using 
      EzColocalization to digitize the signals of punctum-distributed P-LApr proteins 
      co-localized with phagosomes and to calculate percentages of phagosomes engaged. 
      We showed that SYBR Gold nucleic acid dye could visualize intracellular 
      mycobacteria that did not express a fluorescent protein. This protocol was 
      validated by showing that IFN-gamma enhanced the co-localization of a 
      punctum-distributed P-LApr protein (LC3) with Mycobacterium bovis BCG in the 
      monocyte/macrophage-like RAW264.7 cells and that there was greater 
      co-localization of LC3 with BCG than with M. tuberculosis H37Rv in bone 
      marrow-derived macrophages (BMDMs). Although BCG and a derived strain (rBCG-PA) 
      showed a similarly high degree co-localization with LC3 in BMDMs, in RAW264.7 
      cells BCG showed much less co-localization with LC3 than rBCG-PA indicating the 
      need for caution in interpreting biological significance from studies in cell 
      lines.
FAU - Wu, Kang
AU  - Wu K
AD  - Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular 
      Virology of MOE/MOH, Fudan University, Shanghai, 201508, China.
AD  - TB Center, Shanghai Emerging and Re-emerging Institute, Shanghai, 201508, China.
FAU - Yan, Bo
AU  - Yan B
AD  - Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular 
      Virology of MOE/MOH, Fudan University, Shanghai, 201508, China.
FAU - Lowrie, Douglas B
AU  - Lowrie DB
AD  - Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular 
      Virology of MOE/MOH, Fudan University, Shanghai, 201508, China.
AD  - TB Center, Shanghai Emerging and Re-emerging Institute, Shanghai, 201508, China.
FAU - Li, Tao
AU  - Li T
AD  - Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular 
      Virology of MOE/MOH, Fudan University, Shanghai, 201508, China. litaokc@126.com.
AD  - TB Center, Shanghai Emerging and Re-emerging Institute, Shanghai, 201508, China. 
      litaokc@126.com.
FAU - Fan, Xiao-Yong
AU  - Fan XY
AD  - Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular 
      Virology of MOE/MOH, Fudan University, Shanghai, 201508, China. 
      xyfan008@fudan.edu.cn.
AD  - TB Center, Shanghai Emerging and Re-emerging Institute, Shanghai, 201508, China. 
      xyfan008@fudan.edu.cn.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20210113
PL  - England
TA  - Sci Rep
JT  - Scientific reports
JID - 101563288
SB  - IM
MH  - Animals
MH  - Fluorescent Antibody Technique
MH  - Image Processing, Computer-Assisted
MH  - Lysosomes/*microbiology/pathology
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - Mice, Inbred C57BL
MH  - Mycobacterium/*isolation & purification
MH  - Mycobacterium Infections/microbiology/*pathology
MH  - Phagosomes/*microbiology/pathology
MH  - RAW 264.7 Cells
PMC - PMC7807018
COIS- The authors declare no competing interests.
EDAT- 2021/01/15 06:00
MHDA- 2021/08/17 06:00
PMCR- 2021/01/13
CRDT- 2021/01/14 05:38
PHST- 2020/08/12 00:00 [received]
PHST- 2020/11/30 00:00 [accepted]
PHST- 2021/01/14 05:38 [entrez]
PHST- 2021/01/15 06:00 [pubmed]
PHST- 2021/08/17 06:00 [medline]
PHST- 2021/01/13 00:00 [pmc-release]
AID - 10.1038/s41598-020-79425-5 [pii]
AID - 79425 [pii]
AID - 10.1038/s41598-020-79425-5 [doi]
PST - epublish
SO  - Sci Rep. 2021 Jan 13;11(1):1096. doi: 10.1038/s41598-020-79425-5.