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

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 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.