RPB1 immunofluorescence in HEK293 ARMC5 knockout cells

Description

HEK293 cells or ARMC5 K.O. cells were fixed in 4% paraformaldehyde (EMS Emgrid 15710) for 15 minutes, then permeabilised in 0.25% Triton X100 (Sigma Aldrich 93443) for 10 minutes. Cells were incubated in 50% blocking buffer (Millenium Biosciences Li-Cor Intercept in PBS, LCR-927-70001) in PBS for 30 minutes, before being stained with primary antibodies in 50% blocking buffer in PBS for 90 minutes. Cells were then incubated for 30 minutes with secondary antibodies plus DAPI at 200 ng/mL in 50% blocking buffer in PBS. imaging was performed on a Nikon Ti2 microscope equipped with a Yokogawa CSU-W1 spinning disk, with 40x/NA0.95 Plan Apo λ air objective, and dual Hamamatsu ORCA-Fusion C14440-20UP cameras. 20 z-planes at 1 µm intervals were acquired. DAPI DNA stain was acquired with a 405 nm laser and 450/82 nm filter. Alexa488+ conjugated secondary antibodies were acquired with a 488 nm laser and 525/50 nm filter, and mCherry-RPB1 was acquired with a 561 nm laser and 617/73 nm filter. Where applicable, Alexa488-NHS or Alexa647-NHS cell stains were acquired with the appropriate green (525/50 nm) or far-red (685/40 nm) filter.

Steps to reproduce

- Folders 20240329_104049_713, etc. contain single-cell quantifications over time (QUANTIFICATION subfolder) and imaging metadata (OME-TIFF-MIP_csv subfolder) - hek_IF_analysis.html shows all data analysis steps from the single-cell data provided and generates PLOTS and SUMMARIES

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