Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

Summary

PeroxiSPY probes are used to detect peroxisomes in mammalian cells. Here we demonstrate how to use the probes to stain peroxisomes in live and fixed cells.

Abstract

Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization of peroxisomes is key to identifying peroxisome dysfunction; however, progress has been limited by the lack of peroxisome-specific probes. By developing PeroxiSPY, peroxisome-specific probes that mimic natural peroxisome metabolites, we enabled real-time detection, tracking, and quantification of peroxisome dynamics in live mammalian cells. Additionally, the applications of this technique to patient cells with peroxisome disorders reveal pathological phenotypes. Here, we provide a detailed protocol for the detection of peroxisomes in live mammalian cells, as well as the detection of peroxisomes post-staining in fixed cells. We demonstrate how to stain peroxisomes in live cells using peroxisome-specific probes, focusing on the preparation of cells, timing of the procedure, reproducibility, and troubleshooting. These probes enable rapid, specific, and non-toxic live-cell detection of peroxisomes, offering a unique, quantitative method for assessing peroxisome function and its dysfunction in peroxisome-related disorders.

Introduction

The peroxisome is the most abundant eukaryotic organelle, with some cells exceeding a thousand peroxisomes¹. Their functions are critical for organism development and metabolic adaptation^1,2, evidenced by a range of human peroxisome disorders, typically incurable neurodegenerative and neurometabolic conditions in young children^3,4. To our knowledge, there has previously been a lack of selective methods available to label peroxisomes in live cells and tissues. These tools are invaluable for research and diagnostic applications, allowing direct and quantitative measurement of several aspects of peroxisome formation and function⁶. These probes are versatile enough to, on one hand, provide a quantitative readout of peroxisome function in cells, and on the other hand, be deployed in translational efficacy assays or as diagnostics for peroxisome disorders⁶. PeroxiSPY staining is a rapid, specific, non-cytotoxic technique for non-invasive live-cell peroxisome detection. Here, we describe how to stain peroxisomes in mammalian cell lines using peroxisome probes (the probes have also demonstrated specific peroxisomal staining in zebrafish embryos, but are not suitable for staining of peroxisomes in plant Arabidopsis thaliana cells⁶). The staining can be achieved in under 10 min. The probe can then be detected for hours or be fixed using a paraformaldehyde fixation protocol for further immunostaining.

Protocol

1. Staining live cells

1. Cell culture preparation for live-cell imaging (duration: 24 h)
   NOTE: The study followed standard cell culturing protocols for all experiments. Cell lines were maintained in DMEM or DMEM/F12 for SH-SY5Y, supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, at 37 °C and 5% CO₂. Some imaging systems are sensitive to the media composition. If the microscope detects the signal from phenol red in the media, use phenol red-free alternatives. Additionally, if the microscope is not equipped with a CO₂ chamber, replace the media with a HEPES (pH 7.4)-containing media.
   1. One day before the experiment, split the cells into a glass-bottom imaging plate to achieve a 30-70% confluency the next day.
      NOTE: In this protocol, 4-well 35 mm plates were used.
      1. (Critical) Do not use starved or stressed cells to split for the experiment. Ensure that the cell lines have fresh media changes regularly (at least every 2-3 days) and are split before they reach 90% confluency. The goal is to have a healthy cell line to ensure a uniform staining.
         NOTE: Some cells, e.g., stem cells, will require glass coating with an appropriate coating layer, e.g., Matrigel, which does not interfere with staining.
2. Preparation of the imaging setup (duration: 1h)
   NOTE: Imaging organelles and organelle dynamics requires high spatial resolution. For this protocol, a confocal microscope equipped with a GaAsP photomultiplier module, a CO₂ chamber, and a temperature control unit was used. The imaging was performed using a bi-directional galvanometer scanning mode with a CFI Plan Apo Lambda 60x oil NA 1.42 objective, and a 561 nm laser (50 mW) was used for the 555 probe. Use a 640 nm laser or equivalent for the 650 probe. Peroxisomes were visualized in human cell lines. The incubation time with the probes may differ for different cell lines. It is recommended to optimize the timing by visualizing cells at different time points after staining (Figure 2A).
   1. Initiate the confocal system. Ensure that the CO₂ and temperature (37 °C) are stabilized for 20 min before starting the staining and introducing the cells to the microscope.
      NOTE: (Critical) It is essential to verify that cells remain healthy throughout the experiment by visual inspection and that the pH and temperature of the media are maintained. This can be achieved by utilizing control units for parameters and visually inspecting the media coloring, if phenol red is used. For that, temperature and CO₂ controllers can be used.
   2. Set up the confocal microscope software. Initiate the software and turn on the 561 nm laser for the 555 probe staining. Adjust the parameters of acquisition and laser power. This protocol uses 1% laser power, HV (gain) of 100, and a resolution of 1024 1024 pixels with 3 zoom to image the cells.
3. Peroxisome staining (Duration: 1-5 min)
   1. Pipette an aliquot of the probe for a final working concentration of 1 µM, e.g., for 0.5 mL of media, pipette 0.5 µL of the probe.
   2. Select the well to stain, and label it on the outside with a marker pen.
   3. From the chosen well, pipette out all (0.5 mL) of the media and dispense it into a microcentrifuge tube containing the probe aliquot from step 1.3.1.
      NOTE: It is critical to remove all media from the well in this step to ensure uniform staining.
   4. Pipette up and down 3 times to mix the media and the probe. Aspirate the total mixture into the pipette.
      NOTE: Mixing by vortexing can be used instead of pipetting.
   5. Dispense the mixture back into the chosen well drop by drop, ensuring the pipette is in contact with the wall of the well. Upon adding the mixture back into the well, start a 10-min timer.
   6. Close the lid, add a drop of immersion oil on the objective (this protocol uses 60× oil objective), and position the plate on the microscope for imaging.
      NOTE: Optimizing the concentration of the probe for each cell line is recommended to ensure a uniform staining. Testing at 0.25, 0.5, 1, and 2 µM for 5-20 min is recommended; lower concentrations typically stain with lower background (Figure 2A). Depending on the cell line and microscope detector sensitivity, stained cells can be imaged from as soon as 5 min after addition of the probe to 24 h after addition. In some cases, for example, imaging of stem cells, visualization of cells can be conducted for over 180 min after probe addition without a change in the signal. At any time point, the media can be changed to wash out the probe. This will lead to a gradual loss of the staining in about 30 min for HEK293T cells.
4. Confocal Imaging of peroxisomes in live cells (duration: 10 min-3 h)
   1. Once the plate is positioned on the microscope, adjust focus on the cells in the well that is stained and start imaging.
   2. Once the cells are in focus, adjust the gain (HV or detector sensitivity) and laser power settings using standard confocal microscope setup guidelines.
   3. Find and position (Zoom-In) onto the group of cells. After focusing on the cells, adjust the parameters of acquisition to the required image quality.
      1. Set the imaging parameters for image acquisition: 0.5-1% laser power, 100 HV (gain) in NIS-Elements Software. Check that the image is not oversaturated (if it is, alter intensity and gain).
      2. Select the scan size. Acquire 1024 1024 px images, scan speed (1/2-1/4), line averaging (2), zoom (3) that is appropriate for the imaging being conducted.
         NOTE: Peroxisomes move during image acquisition, which can result in artefacts. Recommended parameters are optimized to minimize the movement traces while ensuring sufficient quality of the images for quantification. When creating a time-lapse, ensure that interval, duration, and loops are specified before starting the video. The probes are photostable.
   4. Acquire and save all images with accessible labels (here, .nd2 NIS-Elements Nikon Software format is used).

2. Staining fixed cells

1. Cell culture preparation for live-cell imaging (duration: 24 h)
   NOTE: Standard cell culturing protocols were followed for all experiments. Huh7 cells were maintained in DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin, at 37 °C and 5% CO₂.
   1. A day before the experiment, split the cells into a plate with coverslips to achieve a 30-70% confluency the next day.
      NOTE: Seed 50,000 cells/well in 12-well plates with 18 mm coverslips, or 15,000 cells/well in 24-well plates with 13 mm coverslips.
2. Peroxisome staining (Duration: 1-5 min)
   1. Dilute the probe with new media to a final working concentration of 4 µM in an empty light-sensitive microcentrifuge tube, e.g., for 0.5 mL of media, pipette 2 µL of the probe (stock solution: 1 mM).
   2. Select the well to stain and label it on the outside with a marker pen.
   3. From the chosen well, aspirate all (0.5 mL) of the media and replace it with the probe medium from step 2.2.1. Dispense the probe medium into the chosen well drop by drop, ensuring the pipette is in contact with the wall of the well.
   4. Upon adding the mixture back into the well, incubate the cells at 37 °C for 10-15 min.
      NOTE: From this moment, the cells are light-sensitive and should be covered with aluminum foil.
3. Post-staining fixation and imaging peroxisomes in fixed cells
   1. After staining, carefully remove the media and wash the cells twice with phosphate-buffered saline (PBS).
   2. Apply 4% paraformaldehyde (PFA) solution in PBS at room temperature for 20 min.
   3. Carefully remove PFA into a specifically designated waste container. Wash cells once with PBS.
      CAUTION: PFA is toxic and must be disposed of according to institutional regulations.
   4. To proceed with immunofluorescence, follow the steps below
      1. Add 0.5% Triton-X (diluted in PBS) to permeabilize the cells. Leave for 2.5 min.
      2. Carefully remove Triton-X. Wash cells twice, for 10 min with PBS.
      3. Block with 5% bovine serum albumin (BSA) solution in PBS for 20 min at room temperature or overnight at 4 °C.
      4. Proceed with the standard immunofluorescence procedure.

Results

PeroxiSPY probes are engineered to mimic natural substrates of peroxisomes - branched and very long-chain fatty acids⁵. Therefore, staining will result in an adenosine triphosphate (ATP) Binding Cassette Subfamily D (ABCD)-dependent import of the probes from the cytoplasm into peroxisomes^5,6,7. Depending on the cell metabolic state, the ATP-dependent ABCD transport may efficiently and rapidly import the probes, which will result in a high peroxisome to cytoplasm ratio of fluorescence intensity (Figure 1A). Critical steps to ensure a high ratio include avoiding starvation of cells by regularly replacing cell media and not growing cells at high confluency.

To verify the specificity of peroxisome staining, it is recommended to include several controls (Figure 1B,C). Co-localization analysis with a reference peroxisomal marker, for example, protein import marker - GFP-SKL (Figure 1B,C). Additionally, if peroxisome-deficient cell lines are available, e.g., PEX19 KO, the staining will be absent in those cells (Figure 1B)^5,8.

Peroxisome probes can be used to measure peroxisome density (Figure 1C)^5,9, and are being developed to assess substrate import, peroxisome dynamics, and interactions. For quantitative imaging, it is important to standardize the staining procedure, the timing of image acquisition (e.g., 10-20 min after the staining), the cell line preparation, and the parameters of acquisition. Some cell lines require optimization of the probe concentration as well as the timing in order to improve the signal intensity of the peroxisomes relative to the cytosolic background, e.g., 250 nM and 20 min work better for COS-7 cells (Figure 2A). Cells metabolize the probes, which leads to a gradual reduction of peroxisome staining over time; however the ratio of peroxisome to cytoplasm can improve over 24 h (Figure 2B). Additionally, we developed a fixation procedure, which allows co-staining with antibodies and visualization of peroxisomes at a later time (Figure 2C,D).

Figure 1: Live cell imaging of peroxisomes. (A) Schematic of staining. (B) Confocal microscopy of WT and PEX19 KO HEK293T cells overexpressing GFP-SKL and stained with the red peroxisome probe (1 µM for 10 min). Scale bars - 1 µm and 10 µm. The representative intensity profile is shown.(C) Confocal microscopy of human fibroblasts overexpressing GFP-SKL and stained with PeroxiSPY (1 µM for 10 min). Scale bars - 1 µm and 10 µm. The representative intensity profile is shown.(D) Confocal microscopy of SW13 (Adrenal Cortex), SH-SY5Y (Neuroblasts), CCL-136 (Myocytes), HaCat (Keratinocytes), U2OS (Osteoblasts), and HEK293T (Human Embryonic Kidney) cells stained with the red peroxisome probe (1 µM for 10 min). Scale bars - 2 µm and 10 µm. Quantification was conducted using Fiji as previously described⁹ and shows the number of peroxisomes per micron square, mean ± SEM, N = 100 cells pooled from 3 biological repeats, *** - p < 0.001, **** - p < 0.0001, Kruskal-Wallis Test. Please click here to view a larger version of this figure.

Figure 2: Peroxisome imaging optimization and staining of fixed cells. (A) Confocal microscopy of COS-7 (African green monkey kidney) cells stained with the red peroxisome probe according to the protocol (1 µM for 10 min; before optimization) and under optimized conditions (250 nM for 20 min; after optimization). Scale bar - 10 µm. Mean pixel intensity of peroxisomes and cytosol was quantified using Fiji and expressed as a ratio. N = 7 cells (before optimization) and 21 cells (after optimization). Mean ± SEM, *** - p < 0.001, **** - p < 0.0001, Shapiro-Wilks test. (B) Confocal microscopy of HEK293T cells stained with the red peroxisome probe (1 µM) for 15 min and 24 h. Quantification shows fluorescence intensity of peroxisomes and cytoplasm, as well as the ratio of peroxisome to cytoplasm, mean ± SEM, N = 680 peroxisomes pooled from 3 biological repeats, **** - p < 0.0001, Shapiro-Wilks test. (C) Confocal microscopy of Huh7 cells stained with peroxisome probes using the fixation protocol. Peroxisomes were co-stained with anti-PEX14 primary antibody (1:100 in blocking solution, 1 h incubation at RT) and secondary antibody (1:1000 in blocking solution, Goat anti-Rabbit Alexa Fluor 488, 1 h incubation at RT). Representative confocal images are shown. Scale bars - 10 µm. (D) Quantification shows the changes in peroxisome fluorescence intensity during fixation and permeabilization that was performed on HEK293T cells according to the protocol, mean ± SEM, N = 100 cells pooled from three biological repeats, **** - p < 0.0001, Shapiro-Wilks test. Please click here to view a larger version of this figure.

Discussion

This simple peroxisome detection can be done in under 10 minutes in animal cells. PeroxiSPY staining allows for visualization, quantification, and tracing of peroxisomes in live cells and is available as a red (Table of Materials) and a far-red probe⁵ (Table of Materials). The staining provides a similar peroxisome visualization to a classical peroxisome targeting signal (PTS1)l-based staining¹⁰ (Figure 1B) without the need for overexpression. The fluorophores are photostable and can be used for super-resolution imaging and long-term acquisition of peroxisome dynamics in different experimental conditions^5,9,11. Additionally, the probes are sensitive to peroxisome functioning; therefore they can be used to detect and quantify peroxisome pathology⁵ for research and diagnostic applications. Peroxisome staining depends on peroxisome metabolism and the capacity of peroxisomes to import and oxidize fatty acids. It is, therefore, important to optimize the concentration and the timing for the cell lines used. Here we present a standard procedure that works for the cell lines we tested; however timing of staining can be extended to 24 h without the loss of peroxisome-to-cytoplasm fluorescence intensity ratio (Figure 2B).

The probes were tested in many mammalian cell lines (Figure 1 and Figure 2) and in Zebrafish Danio rerio cells and embryos⁵, displaying specificity to peroxisomes and ease of use. These probes were not tested in other animal cell lines, and the conditions, as well as probe concentrations, will need to be optimized for them. The probes do not stain peroxisomes in plant Arabidopsis thaliana cells⁵.

Some problems and their respective solutions for troubleshooting are discussed here. If the peroxisome to cytoplasm ratio is too low (low peroxisome staining), make sure that cells are freshly split and then split them again for the staining. Use 30-70% confluency for staining. Optimize the concentration of the probe (too high concentration will lead to exceeding the peroxisome capacity to uptake the probes) and the timing (for example, see Figure 2A). Use a 35 mm glass-bottom dish (staining in 96-well plates is more challenging due to the small volume mixing). Ensure that the probes are added to the microcentrifuge tube and thoroughly mixed with the media before addition to the cells. Different cell lines and varying conditions will have an effect on staining. Finally, washing out the probe will reduce the background; however, the signal will be gradually lost, reducing the time in which imaging can be conducted effectively.

If there are no visible peroxisomes, ensure that the probe was mixed with the media. Increase the concentration of the probe (e.g., to 2 µM). Introduce an additional peroxisomal marker to ensure that peroxisomes can be visualized. If there is cell-to-cell variation in staining, it is important to remove all the media and mix it with the aliquot thoroughly before adding it back to the cells. This ensures uniform staining. If there is still variation, it is possible that the population of cells is heterogeneous in their peroxisome substrate import capacity. Synchronization of cells by regular splitting aids in uniform staining. If no signal is detected, check that the laser settings on the microscopes are compatible with the probe excitation/emission parameters. If the background outside of the cell is too bright, wash out the probes to remove this background; however, the signal will be gradually lost, reducing the window of time in which imaging can be conducted effectively. Additionally, fixation can be used to reduce the background. If the signal post-fixation is very weak, check if the signal is lost after Triton-X 100 addition. This step can be modified or removed depending on the downstream application. Reduce washing steps.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-PEX14 antibody	Proteintech	10594-1-AP
Confocal Microscope	Nikon	A1r	Any confocal microscope with a high NA objective, e.g. 60x oil NA1.4
DMEM	Pan Biotech	P04-03590	cell culture media
Glass-bottom imaging plates	Cellvis	D35C4-20-1.5-N
Goat anti-Rabbit Alexa Fluor 488	ABCAM	ab150081
HEK293T	ATCC	CRL-3216
PeroxiSPY555	Spirochrome	https://spirochrome.com/product/peroxi_spy555/	https://doi.org/10.1038/s41467-024-48679-2
PeroxisPY650	Spirochrome	https://spirochrome.com/product/peroxi_spy650/	https://doi.org/10.1038/s41467-024-48679-2