# Protocol for immunofluorescence detection and quantitative analysis of pH-dependent transcriptional condensates

## TL;DR

This paper provides a detailed protocol for imaging and analyzing pH-dependent transcriptional condensates in macrophages using advanced microscopy and image processing tools.

## Contribution

The novel contribution is a comprehensive and reproducible protocol for pH-controlled immunofluorescence and quantitative analysis of BRD4 condensates.

## Key findings

- Acidic pH regulates BRD4 and MED1 condensate assembly in mouse and human cells.
- A protocol is provided for imaging and quantifying BRD4 condensates in macrophages using confocal and STED microscopy.
- Image processing pipelines using FIJI, CellProfiler, and MATLAB are detailed for condensate analysis.

## 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

•Steps for pH adjustment of macrophage growth medium•Instructions for pH treatment and immunofluorescence staining of BMDMs•Details on the use of CellProfiler for condensates counting across pH conditions•Details on the use of MATLAB for quantitative analysis of condensates properties

Steps for pH adjustment of macrophage growth medium

Instructions for pH treatment and immunofluorescence staining of BMDMs

Details on the use of CellProfiler for condensates counting across pH conditions

Details on the use of MATLAB for quantitative analysis of condensates properties

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.