Protocol for evaluating compound uptake and RNase L co-localization in live cells using fluorescence-based binding, competition assay, and confocal microscopy

Summary

Using ribonuclease targeting chimera (RIBOTAC) technology, fluorescent probes enable real-time visualization of RNase L localization and interaction dynamics. Here, we present a protocol to assess probe uptake, binding specificity, and RNase L co-localization in live cells using a fluorescent-based binding and competition assay combined with confocal microscopy. We provide step-by-step instructions for live-cell imaging and quantitative fluorescence analysis, enabling researchers to monitor RNA degradation pathways and evaluate the effects of RNA-targeting small molecules with high spatial resolution.

For complete details on the use and execution of this protocol, please refer to Khaskia et al.¹

Graphical abstract

Highlights

• •Fluorescence-based assays to assess probe specificity and RNase L engagement
• •Live imaging of RNase L localization and dynamics using fluorescent probes
• •Quantitative fluorescence analysis provides insights into RNA degradation pathways

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Using ribonuclease targeting chimera (RIBOTAC) technology, fluorescent probes enable real-time visualization of RNase L localization and interaction dynamics. Here, we present a protocol to assess probe uptake, binding specificity, and RNase L co-localization in live cells using a fluorescent-based binding and competition assay combined with confocal microscopy. We provide step-by-step instructions for live-cell imaging and quantitative fluorescence analysis, enabling researchers to monitor RNA degradation pathways and evaluate the effects of RNA-targeting small molecules with high spatial resolution.

Before you begin

Cell culture

Timing: 1–7 days before the experiment

Note: MCF-7 and MDA-MB-231 cell lines were used for our experiments.

Note: For imaging, cells should be cultured on a μ-Slide 8-well plate, a chambered coverslip with 8 wells for cell culture, immunofluorescence, and high-end microscopy.

• 1.
  Maintain both cell lines at 37°C with 5% CO₂.

  • a.
    Culture MCF-7 cells in DMEM medium.
  • b.
    Culture MDA-MB-231 cells in RPMI 1640 medium.
  • c.
    The typical seeding density is 2.2 x 10⁶ cells for 10 cm dishes, 10 x 10⁴ cells for 96-well plates, and 5-11 x 10⁴ cells for μ-Slide chambers.
  • d.
    Cells used for tests are typically within 2 months of culture and <30 passages.

Note: RNase L co-localization requires transfection with an appropriate GFP-tagged RNase L plasmid. Refer to Key Resources Table and to materials and equipment setup for details.

Note: DMEM and RPMI media need to be prepared. Refer to the materials and equipment setup for details.

μ-Slide chamber coating

Timing: 1.5 h

• 2.To prepare a solution of 10% poly-D-lysine (PDL), weigh 1 g of PDL and dissolve in 10 mL of DDW. Stir thoroughly and store at −20°C.
• 3.In a biological hood, add 300 μL of 10% PDL to each well of the μ-Slide 8-well plate.
• 4.Incubate at 37°C for 1 hour.
• 5.Remove PDL coating solution by gentle aspiration.
• 6.To wash, add 300 μL of 1X PBS to each well. Let the slide sit for 5 minutes, gently tilting it a few times during this period. Then decant the PBS. Repeat this wash step two more times (three washes total).

Note: If the slide was stored at 4°C, it should be taken out 30 minutes before cell seeding to allow it dry.

CRITICAL: PDL treated μ-Slides are ready to use immediately after washing with 1X PBS or can be stored at 4°C for a maximum of 24 hours.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Experimental models: Cell lines
MCF-7	ATCC	HTB-22
MDA-MB-231	ATCC	HTB-26
Chemicals, peptides, and recombinant proteins
NaCl	Bio-Lab LTD	0010011077
HCl 37%	Bio-Lab LTD	UN1798
NaOH	Bio-Lab LTD	UN1823
Na2HPO4	Merck	F1544986
EDTA	Fisher Chemical	2191646
Cy5	This study	N/A
Ribo1-Cy5	This study	N/A
Ribo2-Cy5	This study	N/A
Ribo1	This study	N/A
Ribo2	This study	N/A
DMEM	Diagnovum	D7594
RPMI 1640 Medium	Diagnovum	D6481
FBS	Sigma-Aldrich	F9665
PBS	Thermo Fisher Scientific	10010023
Poly-D-lysine	Corning	354210
Trypan blue solution 0.4%	Logos Biosystems	T13001
Trypsin-EDTA 0.25%	Diagnovum	D7225
Penicillin/streptomycin	Diagnovum	D910
DMSO cell culture	Sigma-Aldrich	BCCJ3891
HEPES	Capricorn Scientific	HEP-B
PEI transfection reagent	MedChemExpress	HY-K2014
Opti-MEM	Life Technologies	31985-062
GFP-tagged RNase L plasmid	GeneCopoeia, Inc	EX-U1047-M29
DAPI	Angene Chemical	AG002W8S
RNase L	GenScript	U282HCNSG0-8/P2IH001
Software and algorithms
Fiji ImageJ	Open source	https://imagej.net/ij/download.html
Gen5	BioTek	https://www.agilent.com/en/support/biotek-software-releases
NIS-Elements Viewer	Nikon Instruments Inc	https://www.microscope.healthcare.nikon.com/products/software/nis-elements/software-resources
GraphPad Prism 10	Dotmatics	https://www.graphpad.com/features
Other
10 cm dishes	Corning	430167
96-well plate	Wuxi NEST Biotechnology Co., Ltd	701001
μ-Slide chambers 8-well plate	ibidi GmbH	80826
Synergy H1 hybrid	BioTek, Agilent	N/A
pH meter	MRC laboratory instruments	INE-PHS-3E
Cell counting slides	Logos Biosystems	L12001
Microscope with GFP filter	Evident Olympus	CKX53
Nikon New AX-R confocal microscope	Nikon Corporation Healthcare Business	N/A
Cell counter	Logos Biosystems	L40002

Materials and equipment

RNase L binding buffer preparation

Reagent	Final concentration	Amount
Disodium phosphate (Na2HPO4)	8 mM	113.56 mg
Sodium chloride (NaCl)	0.185 mM	1 mg
EDTA	1 mM	37.2 mg
ddH2O	N/A	100 mL
Total	N/A	∼100 mL

Storage conditions: Store at −20°C for up to 3 months.

CRITICAL: Stir continuously until all compounds are fully dissolved. Adjust the pH to 7.0 using 6 N HCl if the solution is too basic, or 6 N NaOH if it is too acidic.

DMEM cell culture medium

Reagent	Final concentration	Amount
DMEM + L-glutamine	89%	445 mL
FBS	10%	50 mL
Penicillin-Streptomycin	1%	5 mL
Total	100%	500 mL

Storage conditions: Store at 4°C for up to 3 months.

RPMI cell culture medium

Reagent	Final concentration	Amount
RPMI 1640 + L-glutamine	86.5%	432.5 mL
FBS	10%	50 mL
Penicillin-Streptomycin	1%	5 mL
HEPES	2.5%	12.5
Total	100%	500 mL

Storage conditions: Store at 4°C for up to 3 months.

Stock solutions

• •To prepare a 10 mM stock solution, dissolve 1.17 mg of lyophilized Cy5 probe powder in 250 μL of DMSO.
• •To prepare a 10 mM stock solution, dissolve 2.19 mg of lyophilized Ribo1-Cy5 probe powder in 250 μL of DMSO.
• •To prepare a 10 mM stock solution, dissolve 2.23 mg of lyophilized Ribo2-Cy5 probe powder in 250 μL of DMSO.
• •To prepare a 10 mM stock solution, dissolve 1 mg of lyophilized Ribo1 probe powder in 250 μL of DMSO.
• •To prepare a 10 mM stock solution, dissolve 1.1 mg of lyophilized Ribo2 probe powder in 250 μL of DMSO.

Note: Ribo1, Ribo2, and the corresponding fluorescent probes were synthesized in the laboratory following protocols described in our original research publication and are not commercially available.

CRITICAL: Stock solutions should be protected from light to prevent photobleaching.

• •Storage recommendation: Store stock solutions at −20°C, where they remain stable for several months. Aliquoting into 1 mM or 100 μM portions is advised if smaller working concentrations are needed.

Plasmid

• •GFP-tagged RNase L plasmid was obtained from GeneCopoeia (OmicsLink Expression Clone, catalog no. EX-U1047-M29), which encodes human RNase L tagged with GFP. The gene corresponds to NCBI RefSeq accession number GenBank: NM_021133, and the vector backbone is pEZ-M29. The total plasmid size is 8,390 bp, and it carries an ampicillin resistance marker for selection. The fusion protein includes an N-terminal GFP tag for visualization.

Other solutions used in this protocol

• •A poly-D-lysine (PDL) solution can be prepared by dissolving PDL powder in DDW. The typical concentration of PDL standard solution for slide coating is 10% w/v. A stock solution of 1 mg/mL can be prepared by dissolving 1 mg of PDL powder in 1 mL of DDW and then stirring thoroughly.
• •RNase L-GST protein was purchased from GenScript at a stock concentration of 0.2 mg/mL. A 10 nM stock concentration was prepared in RNase L buffer for binding and competition assays.
• •DAPI solution can be prepared by dissolving DAPI powder in PBS. The typical concentration of DAPI standard solution for live-cell imaging is between 1-5 μg/mL based on the cell line. A stock solution of 1 mg/mL can be prepared by dissolving 1 mg of DAPI powder in 1 mL of PBS and then stirring thoroughly.
• •6 N HCl can be prepared by mixing 49.8 mL of concentrated HCl (37%, ∼12.1 N) with 50.2 mL of distilled water in a fume hood to reach a final volume of 100 mL.
• •6 N NaOH can be prepared by dissolving 24.0 g of NaOH pellets in distilled water and adjusting the final volume to 100 mL.

CRITICAL: The DAPI stock solution should be protected from light to prevent photobleaching.

CRITICAL: Sodium hydroxide is hazardous; it is corrosive to metals and can cause severe skin burns and eye damage. Use appropriate precautions, including protective gloves, lab coat, safety goggles, and face protection. Always add acid to water, not water to acid, to prevent splashing and exothermic reactions. Perform all preparations in a chemical fume hood.

• •Storage recommendation: Keep stock solutions of PDL and DAPI at −20 °C, where they remain stable for several months. RNase L stock and working solutions should be stored at −80 °C, where they remain stable for several months.

Microscope setup

• •Power on the confocal microscope following the manufacturer’s guidelines and allow the laser light source to warm up.
• •Select the appropriate objective, typically a 60x or 100x oil-immersion lens, depending on the target, filter configurations, and detector settings. The commonly used channels include blue (DAPI) – 405 nm excitation and 418–495 nm emission, green (GFP) – 488 nm excitation and 499–550 nm emission, and far-red (Cy5) – 640 nm excitation and 650–750 nm emission.
• •Place a drop of the suitable immersion oil onto the objective before imaging.

Step-by-step method details

Fluorescence-based binding assay

Timing: 3 h

This step measures the interaction between a fluorescently labeled ligand and its target protein by detecting changes in fluorescence intensity upon binding. These changes arise because the fluorophore’s local environment is altered when the ligand binds to the protein. Depending on the nature of the interaction, some fluorophores may become quenched due to solvent exposure, while others may exhibit increased fluorescence as a result of reduced quenching or restricted rotational freedom. These shifts in fluorescence signal enable quantification of binding affinity and detection of conformational changes, providing insights into molecular interactions.

• 1.Prepare a 10 nM RNase L solution by diluting the stock in binding buffer, ensuring thorough mixing for uniformity.
• 2.Heat RNase L at 95°C for 5 minutes using a heat block or water bath to facilitate proper folding.
• 3.Cool on ice for 5 minutes to stabilize the protein before further handling.
• 4.Add 25 μL of RNase L binding buffer to each well of a 96-well plate, except for the first column, to serve as a diluent.
• 5.Add 50 μL of folded RNase L to the first column, which will contain the highest concentration before dilution.
• 6.Perform a 25 μL serial dilution by transferring 25 μL from the first well (A1) to the next (A2), mixing thoroughly, and repeating sequentially down to the last well (A12). Change tips between transfers to prevent cross-contamination. See Figure 1.
• 7.Add 25 μL of a 40 μM solution of the compound (Ribo1-Cy5, Ribo2-Cy5, or Cy5) to each well.
• 8.Gently mix using an infinity (∞) motion, pipetting, or a plate reader to ensure uniform distribution.
• 9.Incubate at 37°C for 2 hours to allow binding interactions to occur.
• 10.Acquire data using a plate reader and analyze results in GraphPad Prism.

Note: Always include appropriate controls. Increasing concentrations of RNase L serve as an internal positive control, generating a dose-dependent fluorescence signal that confirms specific probe binding and assay sensitivity. Negative controls should include the probe alone (to measure baseline fluorescence), RNase L alone (to assess protein autofluorescence), and a non-binding analog such as Cy5 (to ensure that fluorescence changes result from specific binding and not from the fluorophore or probe scaffold).

Note: A triplicate serial dilution was performed for each compound.

Note: Final volume is 50 μL in each well.

CRITICAL: Due to serial dilution, the compound will be diluted 2-fold; therefore, it should be prepared at double the desired final concentration.

Figure 1.

Serial dilution procedure in a 96-well plate

This schematic illustrates the step-by-step process of performing a serial dilution in a 96-well plate. The first well (A1) contains the highest concentration of the sample (dark green). Serial dilutions are performed by transferring 25 μL from one well to the next (A1 → A2 → A3 … A12) while mixing with the buffer. The concentration decreases progressively along the row, as indicated by the gradient color transition from dark to light green.

Fluorescence-based competition assay

Timing: 3 h

This step assesses the competitive binding of fluorescently labeled compounds by introducing increasing concentrations of unlabeled parental binders and monitoring changes in fluorescence intensity. As the unlabeled compound displaces the fluorescent probe from the target protein, alterations in the fluorescence signal reflect competition at the binding site. This enables evaluation of binding specificity, affinity, and target engagement, providing mechanistic insights into molecular interactions. See Figure 2.

• 11.Prepare a 6 nM RNase L solution by diluting the stock in binding buffer to the required concentration. Ensure thorough mixing to maintain uniformity.
• 12.Heat RNase L at 95°C for 5 minutes to facilitate proper folding. Use a heat block or water bath set to the specified temperature.
• 13.Immediately cool on ice for 5 minutes to prevent protein aggregation and stabilize its conformation before proceeding.
• 14.Add 25 μL of RNase L to each well of a 96-well plate.
• 15.Add 25 μL of the parental binder (Ribo1 or Ribo2) at increasing concentrations (0, 0.1, 1, 10, and 100 μM) into designated wells, maintaining triplicate wells for each concentration. Carefully mix each well to ensure homogeneity without introducing bubbles, which could interfere with fluorescence readings.
• 16.Incubate at 37°C for 10 minutes to reach sufficient binding equilibrium between RNase L and the parental binder. During incubation, ensure the plate remains covered to prevent evaporation.
• 17.Add 25 μL of a 60 μM solution of the fluorescently labeled compounds (Ribo1-Cy5, Ribo2-Cy5, or Cy5) to the wells, ensuring precise pipetting to avoid variability in final concentrations. Gently mix without creating bubbles to maintain accurate fluorescence measurements.
• 18.Incubate at 37°C for 2 hours to allow binding interactions to occur.
• 19.Acquire data using a plate reader and analyze results in GraphPad Prism.

Note: Always include appropriate controls. Increasing concentrations of a parental (unlabeled) binder serve as an internal positive control in competition assays, displacing the fluorescent probe and confirming specific, concentration-dependent competition. Negative controls should include the fluorescent probe alone (to establish baseline signal), RNase L alone (to assess autofluorescence), and a non-binding competitor (to verify that displacement is due to specific binding rather than nonspecific interference).

Note: Final volume is 75 μL in each well.

CRITICAL: All components in this assay will undergo a 3-fold dilution; therefore, they should be prepared at three times the desired final concentration. This means, for example, that RNase L should be prepared at 6 nM to achieve a final concentration of 2 nM after dilution.

Figure 2.

This schematic represents the fluorescence-based competition assay workflow and plate layout

(A) illustrates the assay principle and (B) shows the experimental setup in a 96-well plate.

Confocal microscopy

Timing: 3–4 days

This step evaluates compound uptake and co-localization with RNase L using confocal microscopy, providing insights into intracellular distribution and target engagement. Prior to imaging, cells are transfected with a GFP-tagged RNase L construct to ensure sufficient protein expression for visualization, enabling accurate assessment of co-localization patterns.

• 20.Seed 1.0 x 10⁵ cells per well in a 96-well plate 24 hours before transfection in 100 μL of complete medium and incubate at 37°C with 5% CO₂.
• 21.The following morning, mix 0.5 μg of plasmid DNA with 8.69 μL of Opti-MEM in a sterile tube.
• 22.In a separate sterile tube, prepare the PEI transfection solution by adding 0.42 μL of PEI (1:5 DNA-to-PEI ratio) to 9.58 μL of Opti-MEM and mix gently.
• 23.Add the PEI solution to the DNA solution, pipetting gently to mix.
• 24.Incubate the mixture at 25°C for 20 minutes to allow complex formation.
• 25.Ensure the cells reach at least 80% confluency, then distribute 10 μL of the transfection mixture into each well containing 100 μL of the medium.
• 26.Incubate the cells for 24 hours at 37°C with 5% CO₂.

Note: The volumes and amounts of PEI, DNA, and Opti-MEM provided are optimized for two wells of a 96-well plate for technical consistency.

CRITICAL: 24 hours post-transfection, examine the cells under a microscope to assess the transfection efficiency. If fewer than 60% of the cells are transfected, the transfection should be repeated.

• 27.Wash the cells gently with 1X PBS to remove any residual media.
• 28.Add 0.25% trypsin to detach the cells and incubate at 37°C for 2 minutes. Once detachment is complete, centrifuge the cell suspension at 200 × g for 5 minutes, discard the supernatant, and resuspend the pellet in fresh medium.
• 29.Seed the cells into a PDL-treated μ-Slide at a density of 5–11 x 10⁴ cells per well, ensuring even distribution.
• 30.Incubate the cells at 37 °C with 5% CO₂ for 24 hours.
• 31.Treat the cells with 0.1 μM of the fluorescent probes.
• 32.Incubate the cells for 0, 2, and 24 hours (depending on your time-course design).
• 33.Wash the cells three times with 1X PBS, for 5 minutes each time.
• 34.Keep the cells in 1X PBS and proceed to confocal imaging.

Note: Always include appropriate controls. Cells expressing GFP-tagged RNase L and treated with a validated probe (e.g., Ribo1-Cy5 or Ribo2-Cy5) serve as an internal positive control to confirm specific co-localization. Negative controls should include cells expressing GFP-RNase L without probe (to assess background GFP signal), cells treated with probe alone without RNase L expression (to evaluate nonspecific probe uptake), and cells treated with a non-binding analog (e.g., Cy5) to confirm that observed co-localization is due to specific probe-target interaction.

Alternatives: As an additional positive control, cells can be transfected with poly(I:C), a synthetic dsRNA analog that activates endogenous 2–5A production and RNase L dimerization. This activation enhances RNase L redistribution and can promote stronger or altered co-localization with validated probes, providing a functional benchmark for imaging specificity.

Data processing: Imaging data processing

Timing: ∼30–60 min per dataset

This section describes quantifying the co-localization of fluorescent probes with RNase L using FIJI (ImageJ), following image acquisition via confocal microscopy.

• 35.
  Open the image in FIJI.

  • a.
    Launch FIJI and open your confocal image file via File > Open.
  • b.
    Ensure the image is in a multi-channel format (e.g., RGB, .lif, .nd2, .tif).
• 36.
  Split channels.

  • a.
    Go to Image > Color > Split Channels.
  • b.
    Identify the individual channels (e.g., GFP for RNase L, Cy5 or other for probe).
• 37.
  Set scale (optional).

  • a.
    If your image metadata is not retained, set the correct scale via Analyze > Set Scale.
• 38.
  Select region of interest (ROI).

  • a.
    Use the Rectangle, Freehand, or Polygon selection tools to select the ROI if needed.
  • b.
    Save your ROI via Analyze > Tools > ROI Manager (optional but recommended for consistency).
• 39.
  Background subtraction (optional but recommended).

  • a.
    For each channel, go to Process > Subtract Background.
  • b.
    Use a rolling ball radius (e.g., 50 pixels) depending on image size.
• 40.
  Co-localization analysis using Coloc 2.

  • a.
    Go to Plugins > Colocalization > Coloc 2.
  • b.
    In the dialog box:

    • i.
      Channel 1: Select RNase L (e.g., GFP).
    • ii.
      Channel 2: Select probe signal.
    • iii.
      ROI: Use current selection if applicable.
    • iv.
      Choose “Pearson’s correlation coefficient” and check “Display images” and “Save results”.
  • c.
    Click OK to run the analysis.
• 41.
  Interpret the results.

  • a.
    The Coloc 2 output will display:

    • i.
      Pearson’s R value: Degree of linear correlation between channels (ranges from −1 to +1).
    • ii.
      Manders’ coefficients (M1 and M2): Fraction of one fluorophore overlapping with the other.
    • iii.
      Scatterplot and thresholded images to visualize overlap.
  • b.
    A Pearson’s R above 0.5 typically indicates moderate-to-strong co-localization.
• 42.
  Save the output.

  • a.
    Save all result tables and images (File > Save As).
  • b.
    Export graphs or processed overlays for inclusion in figures.

Note: Always include appropriate negative controls (e.g., probe in non-transfected cells) to validate specificity of co-localization.

Expected outcomes

The fluorescence-based binding and competition assays are expected to reveal measurable changes in the fluorescence signal upon interaction between the fluorescent probe and RNase L. These changes may manifest as either an increase or decrease in fluorescence intensity or polarization, depending on the probe’s fluorophore properties, orientation, or local environment upon binding. Similarly, in competition assays, the displacement of the fluorescent probe by an unlabeled ligand may lead to either signal recovery or quenching. Therefore, changes in fluorescence should be interpreted relative to appropriate controls (e.g., probe-only, protein-only, or non-binding compounds). In parallel, confocal microscopy should show intracellular uptake of the fluorescent probe and cytoplasmic expression of GFP-tagged RNase L. Co-localization is expected in cases of successful target engagement and may vary depending on the time point and compound. Quantitative image analysis using Pearson’s correlation coefficient can provide additional support for probe localization and specificity, with expected values typically ranging from 0.4 to 0.8 under optimized conditions.

Limitations

This protocol combines in vitro binding assays and confocal microscopy to assess interactions between fluorescent probes and RNase L, but there are several limitations to consider. Fluorescence changes observed upon binding or competition are not inherently predictable; depending on the fluorophore’s environment, signal intensity may increase or decrease. This variability highlights the importance of including well-matched controls for reliable interpretation. In cell-based experiments, differences in transfection efficiency, probe uptake, or RNase L expression levels may affect the degree of co-localization observed. It’s also important to note that co-localization reflects spatial proximity, not direct binding, and should be supported by additional biochemical evidence where possible. Furthermore, the fluorescent compounds used in this protocol are light-sensitive and should be protected from prolonged exposure to avoid photobleaching or signal loss, both during handling and imaging. Lastly, some degree of optimization may be required when applying this protocol to different cell types or probe variants.

Troubleshooting

Problem 1

The fluorescent probe signal is too weak or is lost over time (Steps 29–33).

Potential solution

Minimize light exposure during handling and imaging by working in the dark or under dim light, ensure proper storage of probe stocks (e.g., light-protected at −20°C), and avoid repeated freeze-thaw cycles.

Problem 2

High background or nonspecific co-localization in microscopy images (Steps 30–33).

Potential solution

Ensure thorough PBS washes to reduce nonspecific fluorescence, verify that the probe is not aggregating or sticking to cellular debris by including a no-transfection control, and avoid overexpression of RNase L, as high levels may lead to artifacts or non-physiological localization.

Problem 3

No measurable change in fluorescence during binding or competition assays (Steps 1–17).

Potential solution

Binding-related changes in fluorescence may be subtle or bidirectional. Always include appropriate controls: probe-only, protein-only, and a non-binding compound if available, confirm that the protein and the fluorescent probe have not degraded, and try a wider range of concentrations for both the protein and the binding/competing compounds.

Problem 4

Low transfection efficiency or weak GFP signal (Step 24).

Potential solution

To improve expression, ensure that the plasmid DNA used is of high purity and free of endotoxins. The DNA-to-PEI ratio may also require optimization depending on the cell line; adjusting the ratio within a range of 1:3 to 1:6 can help enhance transfection outcomes. Additionally, proper cell confluency at the time of transfection is critical. Cells should be approximately 80% confluent to achieve reliable and efficient uptake of the plasmid.

Problem 5

Image analysis in FIJI shows weak or no co-localization correlation (related to Data Processing section).

Potential solution

First, verify that the image channels were correctly split and aligned before analysis. Misalignment or incorrect channel selection can significantly affect the results. Applying background subtraction—using an appropriate rolling ball radius based on image size—can also enhance the accuracy of Pearson’s correlation coefficients. Finally, ensure that only well-transfected, healthy cells are included in the analysis. Avoid selecting fields that contain debris, dead or dying cells, or uneven illumination, as these can introduce artifacts and reduce the reliability of co-localization measurements.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact: Dr. Raphael Benhamou raphael.benhamou@mail.huji.ac.il.

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Elias Khaskia elias.khaskia@mail.huji.ac.il.

Materials availability

All materials are commercially available except for Ribo1, Ribo2, Ribo1-Cy5, Ribo2-Cy5, and Cy5, which were synthesized in the lab. Information regarding protocols for synthesis, please refer to the original manuscript by Khaskia et al.¹

Data and code availability

All original data provided by this study are available through the lead contact.

Acknowledgments

The authors are funded by the Israel Cancer Research Fund (ICRF, 22-201-RCDA); the Israeli Centers for Research Excellence from the Council for Higher Education; the Israel Ministry of Innovation, Science and Technology (0004943); and the Israel Science Foundation (grant 1925/22). E.K.’s fellowship is funded by the Neubauer Fellows in the Sciences Scholarship.

Author contributions

E.K. performed the experiments. E.K. and R.I.B. wrote the manuscript.

Declaration of interests

R.I.B. is a founder of Renasis Bio and a member of its scientific advisory board.