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Updated: Mar 14, 2026
Quantitative Immunofluorescence to Measure Global Localized Translation
Published on: August 22, 2017
Protocol for immunofluorescence detection and quantitative analysis of pH-dependent transcriptional condensates
Zhongyang Wu1, Krishnan Raghunathan2, Zhe Zhong2
1Division of Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA.
Abstract:
Acidic pH regulates the assembly of transcriptional condensates containing BRD4 and MED1 in a variety of mouse and human cells. Here, we present a protocol to image and quantify BRD4 condensates in bone marrow-derived macrophages. We describe steps for preparing macrophage growth medium at controlled pH levels, performing immunofluorescence experiments, and acquiring images with Airyscan confocal and STED super-resolution microscopy. We detail image processing pipelines to analyze condensate properties using FIJI, CellProfiler, and a custom MATLAB program. For complete details on the use and execution of this protocol, please refer to Wu et al.1.
Insights
This study details a method for visualizing and measuring BRD4 condensates in macrophages. Acidic pH influences these transcriptional condensates, impacting cellular processes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Transcriptional condensates are dynamic nuclear structures regulating gene expression.
- BRD4 and MED1 are key components of these condensates.
- Environmental factors like pH can influence condensate assembly and function.
Purpose of the Study:
- To establish a protocol for imaging and quantifying BRD4 condensates in macrophages.
- To investigate the role of acidic pH in BRD4 condensate formation.
Main Methods:
- Macrophage culture with controlled pH media preparation.
- Immunofluorescence staining for BRD4.
- Confocal and super-resolution microscopy (Airyscan, STED).
- Image analysis using FIJI, CellProfiler, and MATLAB.
Main Results:
- Successful imaging and quantification of BRD4 condensates in macrophages.
- Demonstration of pH-dependent regulation of BRD4 condensate assembly.
- Establishment of a robust imaging and analysis pipeline.
Conclusions:
- The presented protocol enables detailed study of BRD4 condensates.
- Acidic pH is a critical regulator of BRD4 condensate formation in macrophages.
- This work provides a foundation for understanding condensate dynamics in cellular regulation.
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