Abstract
Imaging flow cytometry (IFC) has become a powerful tool for studying the activation of transcriptional factors in heterogeneous cell populations in high-content imaging mode. With considerable interest to the clinical development of IFC, the question becomes how we can accelerate its application to solid tissues. We developed the first IFC-based procedure to quantify the nuclear translocation of interferon regulatory factor (IRF) 3, an important measure of induction of type I interferon antiviral response, in primary human immune cells including in solid tissues. After tissue digestion and protocol optimization by spectral flow cytometry, cell suspension is stained for intracellular IRF3 and acquired by IFC. Image analysis is performed using an optimized nuclear mask and similarity score parameter to correlate the location of IRF3 staining and a nuclear dye. The technique measures IRF3 activation at a single cell level and can detect small changes in the percent of activated cells providing objective quantitative data for statistical analysis.
| Original language | English |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press Inc. |
| Pages | 125-153 |
| Number of pages | 29 |
| DOIs | |
| Publication status | Published - Jan 1 2018 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 1745 |
| ISSN (Print) | 1064-3745 |
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Keywords
- Cellular heterogeneity
- Imaging flow cytometry
- IRF3
- Nucleocytoplasmic translocation
- Signal transduction
- Solid tissue
ASJC Scopus subject areas
- Molecular Biology
- Genetics
Cite this
Quantitation of IRF3 nuclear translocation in heterogeneous cellular populations from cervical tissue using imaging flow cytometry. / Trifonova, Radiana T.; Barteneva, Natalie.
Methods in Molecular Biology. Humana Press Inc., 2018. p. 125-153 (Methods in Molecular Biology; Vol. 1745).Research output: Chapter in Book/Report/Conference proceeding › Chapter
}
TY - CHAP
T1 - Quantitation of IRF3 nuclear translocation in heterogeneous cellular populations from cervical tissue using imaging flow cytometry
AU - Trifonova, Radiana T.
AU - Barteneva, Natalie
PY - 2018/1/1
Y1 - 2018/1/1
N2 - Imaging flow cytometry (IFC) has become a powerful tool for studying the activation of transcriptional factors in heterogeneous cell populations in high-content imaging mode. With considerable interest to the clinical development of IFC, the question becomes how we can accelerate its application to solid tissues. We developed the first IFC-based procedure to quantify the nuclear translocation of interferon regulatory factor (IRF) 3, an important measure of induction of type I interferon antiviral response, in primary human immune cells including in solid tissues. After tissue digestion and protocol optimization by spectral flow cytometry, cell suspension is stained for intracellular IRF3 and acquired by IFC. Image analysis is performed using an optimized nuclear mask and similarity score parameter to correlate the location of IRF3 staining and a nuclear dye. The technique measures IRF3 activation at a single cell level and can detect small changes in the percent of activated cells providing objective quantitative data for statistical analysis.
AB - Imaging flow cytometry (IFC) has become a powerful tool for studying the activation of transcriptional factors in heterogeneous cell populations in high-content imaging mode. With considerable interest to the clinical development of IFC, the question becomes how we can accelerate its application to solid tissues. We developed the first IFC-based procedure to quantify the nuclear translocation of interferon regulatory factor (IRF) 3, an important measure of induction of type I interferon antiviral response, in primary human immune cells including in solid tissues. After tissue digestion and protocol optimization by spectral flow cytometry, cell suspension is stained for intracellular IRF3 and acquired by IFC. Image analysis is performed using an optimized nuclear mask and similarity score parameter to correlate the location of IRF3 staining and a nuclear dye. The technique measures IRF3 activation at a single cell level and can detect small changes in the percent of activated cells providing objective quantitative data for statistical analysis.
KW - Cellular heterogeneity
KW - Imaging flow cytometry
KW - IRF3
KW - Nucleocytoplasmic translocation
KW - Signal transduction
KW - Solid tissue
UR - http://www.scopus.com/inward/record.url?scp=85042559438&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85042559438&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-7680-5_8
DO - 10.1007/978-1-4939-7680-5_8
M3 - Chapter
AN - SCOPUS:85042559438
T3 - Methods in Molecular Biology
SP - 125
EP - 153
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -