dSTORM of actin in fixed HeLa cells
Ezra Bruggeman, ruby peters
Abstract
This is a protocol for the preparation of a HeLa DTS cells for dSTORM imaging of the actin network. This protocol was used to generate the data shown in Figure 4 of the following publication:
- Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479
Steps
Cell culture
HeLa TDS cells were cultured in DMEM (Gibco, Invitrogen) supplemented with 10 % Fetal Bovine Serum (FBS, Life Technologies), 1 % penicillin/streptomycin (Life Technologies), and 1 % glutamine (Life Technologies) at 37°C+ 5 % CO2.
Cells were periodically tested for mycoplasma contamination and passaged 3 times per week.
Cells were plated at low density on high-precision glass coverslips (MatTek, P35G-0.170-14-C) 1 day prior to fixation for dSTORM experiments.
Fluorescent labelling
Simultaneously fix and permeabilize the cells in cytoskeleton buffer (CBS, 10 mM MES, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA, 4.5 % sucrose w/v, pH 7.4) + 4 % paraformaldehyde (PFA) and 0.2 % Triton for 0h 6m 0s at 37°C
Further fix the cells in CBS + 4 % PFA for 14Mass Percent at 37°C.
Wash the cells 3 times in PBST (PBS supplemented with 0.1 % Tween).
Permeabilize a second time in PBS + 0.5 % Triton for 0h 5m 0s at Room temperature.
Wash the cells 3 times in PBST.
Blocked the cells for 0h 30m 0s in 5 % BSA.
Wash the cells 3 times in PBST.
Incubate the cells with Alexa FluorTM 488 Phalloidin (A12379, Invitrogen, 1:50 in PBS) for 1h 0m 0s in the dark.
Wash the cells 3 times in PBS.
Prior to dSTORM imaging, replace the PBS with dSTORM imaging buffer (base buffer consisting of 0.56 M glucose, 50 mM Tris (pH 8.5), and 10 mM NaCl supplemented with 5 U/mL pyranose oxidase (Sigma, P4234), 10 mM cysteamine (Sigma, 30070), 40 µg/mL catalase (Sigma, C100) and 2 mM cyclooctatetraene (Sigma, 138924).
