May 09, 2024

Public workspacedSTORM of actin in fixed HeLa cells

DOI
dx.doi.org/10.17504/protocols.io.q26g714j8gwz/v1
  • 1University of Cambridge
Ezra Bruggeman
Open access
DOI: dx.doi.org/10.17504/protocols.io.q26g714j8gwz/v1
Protocol Citation: Ezra Bruggeman, ruby peters 2024. dSTORM of actin in fixed HeLa cells. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g714j8gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 09, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 99507
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000509
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:

Cell culture
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 Temperature37 °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
Fluorescent labelling
1h 41m
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 Duration00:06:00 at Temperature37 °C

6m
Further fix the cells in CBS + 4 % PFA for Concentration14 Mass Percent at Temperature37 °C .
Wash the cells 3 times in PBST (PBS supplemented with 0.1 % Tween).
Permeabilize a second time in PBS + 0.5 % Triton for Duration00:05:00 at TemperatureRoom temperature .

5m
Wash the cells 3 times in PBST.
Blocked the cells for Duration00:30:00 in 5 % BSA.

30m
Wash the cells 3 times in PBST.
Incubate the cells with Alexa Fluor‱ 488 Phalloidin (A12379, Invitrogen, 1:50 in PBS) for Duration01:00:00 in the dark.

1h
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).