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Artículo de método

Imagen y cuantificación de células inmunitarias en el microambiente tumoral utilizando un modelo de neuroblastoma en pez cebra

141 visualizaciones

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DOI:

10.3791/72028

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18 de septiembre de 2026

 ,  ,  ,  ,  , 
1Departments of Pharmacology and Medicine, Section of Hematology and Medical Oncology, The Cancer Research Center, Boston University School of Medicine, 2Tulane University School of Medicine

* These authors contributed equally

En este artículo

Resumen

Este protocolo describe métodos para generar pececitos cebra transgénicos compuestos, obtener imágenes de células inmunitarias asociadas con tumores en pececitos cebra larvales y adultos, y cuantificar la frecuencia de células inmunitarias dentro del microambiente tumoral.

Resumen

La reclutación de células inmunitarias hacia el microambiente tumoral (TME) es un componente importante de la regulación inmunitaria y la progresión tumoral. El estudio de las relaciones espaciales y temporales entre células inmunitarias y tumorales in vivo puede proporcionar información sobre las interacciones que ocurren dentro del TME. Sin embargo, visualizar y cuantificar estas interacciones en organismos intactos sigue siendo un desafío con los enfoques convencionales de imagen y histología. Los peces cebra constituyen un modelo valioso para la investigación del cáncer debido a su facilidad genética, desarrollo rápido y transparencia óptica durante las etapas tempranas del desarrollo, lo que permite la visualización de células marcadas con fluorescencia in vivo. Aquí presentamos un protocolo para generar, identificar, visualizar y cuantificar células inmunitarias y tumorales marcadas diferencialmente con marcadores fluorescentes en un modelo de pez cebra de neuroblastoma. El protocolo incluye el cruce y cribado de peces cebra transgénicos compuestos, la imagen en vivo de larvas, la preparación y visualización de peces adultos portadores de tumores, y la cuantificación de células inmunitarias que rodean y se encuentran dentro de las regiones tumorales. Mediante líneas transgénicas con reporteros, CD4+ células inmunitarias y MYCNcélulas de neuroblastoma impulsadas por pueden visualizarse y analizarse en diferentes etapas del desarrollo. El flujo de trabajo permite la evaluación de la localización de las células inmunitarias en relación con los tumores tanto en peces larvales como adultos, y puede utilizarse para comparar la abundancia de células inmunitarias entre distintas etapas de la enfermedad. Este protocolo proporciona un enfoque reproducible para la obtención de imágenes y la cuantificación de células inmunitarias dentro del microambiente tumoral (TME) in vivo utilizando peces cebra y podría facilitar estudios de las interacciones inmune-tumor en otros modelos de cáncer.

Introducción

El microambiente tumoral (TME) es un ecosistema compuesto por células tumorales, inmunitarias, fibroblastos y células vasculares1. El TME desempeña un papel esencial en la progresión del cáncer, la respuesta terapéutica y la inmunidad antitumoral. Dentro del TME, las células inmunitarias son reguladores clave del destino tumoral, ya sea participando en respuestas antitumorales o induciendo inmunosupresión2. Comprender el reclutamiento, la distribución y la función de las células inmunitarias dentro del TME es fundamental para elucidar los mecanismos mediante los cuales los tumores evaden la vigilancia inmunitaria. Sin embargo, los enfoques convencionales para estudiar el TME, como la histología de punto final, la citometría de flujo y la secuenciación de células individuales, solo proporcionan imágenes estáticas y a menudo requieren la interrupción de la estructura del tejido, lo que resulta en la pérdida de información espacial y de interacciones célula-célula3,4,5,6,7. Además, estos métodos tienen limitaciones en su capacidad para visualizar y cuantificar células inmunitarias dentro del TME, especialmente durante las primeras etapas del desarrollo tumoral.

Los peces cebra (Danio rerio) son un modelo potente para la investigación del cáncer debido a su bajo costo, desarrollo rápido, transparencia relativa y alta conservación genética8,9. Estas ventajas proporcionan un sistema experimental reproducible y escalable que permite tamaños grandes de muestra y cribado eficiente mediante microscopía en un período corto de tiempo10. Es importante destacar que los peces cebra comparten una conservación inmunológica significativa con los seres humanos, incluyendo componentes clave tanto del sistema inmunitario innato como del adaptativo, lo que hace que los hallazgos sean relevantes para enfermedades humanas11. Estas propiedades son particularmente útiles para estudiar cánceres pediátricos como el neuroblastoma8. En consecuencia, se han desarrollado numerosas líneas de reportero inmunitario y modelos de cáncer en pez cebra11. Por lo tanto, el pez cebra sirve como un modelo valioso in vivo para estudiar las interacciones entre células tumorales e inmunitarias, ofreciendo información sobre cómo la regulación inmunitaria influye en el desarrollo tumoral, especialmente durante las etapas iniciales.

Estudios previos que investigan el MTE en pez cebra han dependido en gran medida de modelos de xenoinjertos tumorales combinados con imágenes de punto final para evaluar la localización de células inmunitarias dentro de los tumores12,13,14,15,16,17. Aunque estos métodos proporcionan información útil, están limitados por su dependencia de imágenes bidimensionales, que no pueden capturar completamente la organización tridimensional del MTE ni las relaciones espaciales entre células tumorales e inmunitarias durante el desarrollo14. Además, los modelos de xenoinjerto tumoral implican el trasplante artificial de células tumorales humanas y pueden no recapitular completamente la iniciación tumoral natural18. Estos modelos también son generalmente a corto plazo y no pueden capturar completamente la progresión tumoral ni los cambios en la relación espacial entre células tumorales e inmunitarias durante el desarrollo14,19. Asimismo, este enfoque ofrece una representación limitada de la organización espacial, el contexto de desarrollo y la relevancia fisiológica del comportamiento de las células inmunitarias dentro del MTE. Recientemente, nosotros y otros investigadores hemos aplicado peces cebra transgénicos compuestos, en los cuales las células tumorales e inmunitarias están marcadas diferencialmente con indicadores fluorescentes, para visualizar células inmunitarias asociadas con tumores20,21. Estos peces transgénicos compuestos permiten el estudio del MTE a lo largo del desarrollo del animal y del tumor, facilitando investigaciones sobre las interacciones entre el sistema inmunitario y el tumor in vivo.

Aquí, presentamos un método que utiliza microscopía confocal para estudiar las células inmunitarias dentro del microentorno tumoral (TME) de un modelo de neuroblastoma en pez cebra. En este enfoque, utilizamos líneas reporteras de pez cebra que marcan las células inmunitarias y una línea de pez cebra genéticamente modificada que sobreexpresa el oncogén humano MYCN bajo el promotor de dopamina-β-hidroxilasa (dβh), lo que resulta en el desarrollo de neuroblastoma que se asemeja a la enfermedad humana20. En este modelo transgénico compuesto, las células tumorales e inmunitarias se marcan diferencialmente con EGFP y mCherry, respectivamente. El promotor dβh está activo en las células de la línea simpaticoadrenal y se ha utilizado en múltiples modelos de pez cebra de neuroblastoma22,23,24,25. Además, utilizamos un promotor Cd4 que induce la fluorescencia de mCherry para visualizar las células CD4+15. Se aplicó la adquisición de imágenes confocales con Z-stacks para visualizar el TME en tres dimensiones en diferentes etapas del desarrollo tumoral, permitiendo evaluar las relaciones espaciales entre las células tumorales e inmunitarias dentro del tejido intacto. Este método permite analizar la organización de las células inmunitarias respecto a las células tumorales durante distintas etapas del desarrollo tumoral.

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Protocolo

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University Chobanian & Avedisian School of Medicine under protocol PROTO202100001. The overall workflow for generation of compound transgenic zebrafish, larval imaging, adult tissue preparation, and image analysis is summarized in Figure 1.

Zebrafish transgenic process diagram; fluorescence microscopy, embryo imaging, cryosection analysis.
Figure 1. Workflow for visualization and quantification of immune cells within the zebrafish tumor microenvironment (TME). Schematic overview of the experimental workflow. Immune-reporter zebrafish were crossed with tumor-prone zebrafish to generate compound transgenic offspring expressing fluorescent markers in both immune cells (red) and tumor cells (green). Embryos were screened by fluorescence microscopy to identify compound transgenic fish. Selected fish were either subjected to live confocal imaging during larval development (<21 days post-fertilization [dpf]) or processed by fixation, cryosectioning, and confocal imaging for fish older than 21 dpf. This workflow enables visualization and quantification of immune cells associated with the TME across multiple developmental stages. Part of the illustration was created using BioRender. Please click here to view a larger version of this figure.

1. Generation of Compound Transgenic Zebrafish Lines Through Breeding

  1. Select adult immune-reporter fish and tumor fish that are at least 3 months old for natural breeding.
    NOTE: For the representative results, Tg(cd4-1:mCherry) heterozygous fish were crossed with Tg(dβh:MYCN; dβh:EGFP) heterozygous fish to differentially label immune cells and tumor cells15,23.
  2. On the evening before breeding, fill a two-chamber mating tank two-thirds full with fish water.
  3. Place a grated top insert into the mating chamber. Use dividers to separate one male from one or two females.
  4. Leave the dividers in place overnight. Add artificial plants or other enrichment items to simulate natural breeding conditions.
    NOTE: A 1:2 male-to-female ratio increases the likelihood of successful spawning when breeding tumor male fish with immune-reporter fish. Do not breed two males with one female, as competition between males may occur.
  5. On the following morning, remove the dividers after the facility lights turn on.
  6. Tilt the grated top chamber slightly using the divider as a wedge to create a shallow water area. Allow the fish to spawn for at least 1 h.
  7. Collect eggs from the bottom of the mating chamber by pouring the water through a fine-mesh net.
  8. Transfer the eggs to a sterile Petri dish containing fish water at a density no greater than 1 egg per 50 mm2 (approximately 200 eggs per 100 mm dish).
  9. Remove dead and unfertilized eggs.
    NOTE: Healthy fertilized eggs are translucent and round. Remove cloudy, white, unfertilized, or deformed eggs.
  10. Double-check the fish to avoid mixing sexes or cross-contaminating transgenic tanks.
  11. Return the males and females to their respective tanks.
  12. Place the embryos in a 28.5°C incubator overnight. Do not exceed the embryo density specified in Step 1.8.
  13. Bleach the embryos on the following morning to remove contaminants.
    1. Prepare a working bleach solution consisting of 100 µL bleach in 200 mL system water. Prepare a neutralization solution consisting of 0.5 g sodium thiosulfate in 150–200 mL system water.
    2. Sort and transfer viable embryos into a mesh-net well insert. Submerge the insert in the bleach solution for 5 min.
    3. Transfer the insert directly into the sodium thiosulfate solution. Incubate for 2 min to neutralize residual bleach.
    4. Rinse the embryos in a final bath of fresh system water. Use a squirt bottle to gently transfer the embryos into clean petri dishes containing fresh system water.
  14. Return the embryos to the incubator after bleaching.
  15. Transfer larvae at 5–7 days post-fertilization (dpf) or later to a larger tank containing 5 ppt rotifer water for rearing.
    1. Prepare 5 ppt rotifer water by mixing 1 part stock rotifer water (15 ppt) with 2 parts zebrafish system water.
    2. Verify that the final salinity is 5 ppt using a conductivity meter before transferring the larvae.
      NOTE: Sorting EGFP+ MYCN larvae at 3–5 dpf facilitates identification of tumor-prone fish before rearing. Sorting MYCN embryos at this stage avoids false-negative identification because EGFP expression in neural crest cells may decrease later in development due to MYCN-induced apoptosis.

2. Screening Zebrafish for Fluorescent Markers

  1. Prepare a 0.4% (w/v) tricaine stock solution using system water.
  2. Transfer the fish to a clean petri dish containing only enough system water to cover the bottom.
  3. Add the tricaine stock solution to achieve a final concentration of approximately 0.005% (50 mg/L) for fish younger than 14 dpf or 0.012% (120 mg/L) for fish older than 14 dpf. Confirm adequate anesthesia by observing little to no voluntary movement.
  4. Remove excess water from the dish once the fish are anesthetized and no longer capable of swimming while maintaining visible opercular or cardiac movement.
  5. Screen the fish for fluorescence. Identify green or red fluorescence in the expected region (e.g., the interrenal gland region in MYCN fish).
  6. Separate the fish into groups using a stiff-bristled sorting tool.
    NOTE: Sort tumor-prone fish as early as possible because MYCN-induced apoptosis during later developmental stages may result in false-negative identification. Sort immune-reporter fish immediately before imaging at the larval stage or between 2 and 4 months of age for adult studies. This approach minimizes handling-associated stress and maximizes survival.
  7. Transfer positive and negative fish to separate tanks containing system water for continued rearing.
    NOTE: Step 3 describes handling and imaging of larvae younger than 21 dpf. Steps 4–6 describe fixation, tissue preparation, imaging, and image analysis of older fish.

3. Larval Live-Mounting and Imaging

  1. Place sorted larvae in a 100 mm petri dish containing tricaine fish water (see Step 2.3 for age-appropriate concentrations).
  2. Transfer the sedated larvae to a glass-bottom imaging dish.
  3. Use only enough solution to cover the larva and maintain sedation.
  4. Prepare 1%–1.5% low-melting agarose in fish water. Heat the agarose solution until fully dissolved.
  5. Allow the agarose to cool for 1–2 min. Maintain the agarose at 30°C using a heat block to prevent solidification prior to mounting.
  6. Position each larva laterally in the imaging dish.
  7. Apply approximately 30–200 µL of agarose over the larva to immobilize it. Use larger volumes for larger larvae and smaller volumes for smaller larvae.
  8. Once the agarose has solidified, add approximately 100 µL of 0.005% tricaine solution over the agarose.
  9. Image the larvae using a confocal microscope.
    1. Use a standard laser-scanning confocal microscope with a resonant scanner. Use a 40× objective with a numerical aperture greater than 1.10.
    2. Set the pinhole to 1.0 Airy unit (AU) for all imaging channels.
    3. Use a 488 nm laser for EGFP excitation and a 594 nm laser for mCherry excitation. Keep laser output below 25 mW to minimize photobleaching and phototoxicity. Adjust detector gain and laser power using a range indicator lookup table (LUT) to avoid pixel saturation and black clipping. Do not exceed a detector gain of 800 V because higher voltages introduce excessive noise.
    4. Use a voxel size of 0.42 × 0.42 × 6.0 µm (x, y, z) at a resolution of 1024 × 1024 pixels and a Z-step interval of 6.0 µm. Acquire optical sections continuously through the full volume of interest, typically spanning 90–120 µm in total imaging depth. Use highly sensitive photodetectors operating in 16-bit acquisition mode.
    5. Acquire images using unidirectional line scanning at a resonant scan frequency of 33.3 kHz (approximately 1.58 µs pixel dwell time). Use 2× or 4× averaging to reduce motion artifacts while limiting laser exposure.
      NOTE: Optimize tricaine exposure to maintain larval survival during imaging. After imaging, add fish water until the imaging dish is approximately half full.
  10. Add fresh fish water to the imaging dish immediately after imaging.
  11. Gently remove the larva from the agarose using a stiff-bristled stick and transfer the larva to a dish containing clean fish water for recovery.
  12. Return the fish to its original tank once normal swimming behavior resumes.
    NOTE: For fish older than 21 dpf, the skin overlying the tumor must be removed by cryosectioning to enable high-resolution confocal imaging.

4. Fish Fixation and Cryosectioning

  1. Euthanize fish in accordance with approved institutional animal care guidelines.
  2. Make a ventral incision extending from the anal pore to the pectoral girdle to expose the abdominal cavity and facilitate fixative penetration into internal tissues.
  3. Transfer the fish using forceps into a 1.5 mL tube containing 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) or a volume of fixative at least 10-fold greater than the tissue volume.
    NOTE: Avoid handling the region containing the tumor to preserve tissue integrity.
  4. Place the samples on a nutating rocker set to gentle continuous agitation (30 rpm) and incubate overnight for at least 12 h at 4°C.
    CAUTION: PFA is hazardous, toxic, and potentially carcinogenic. Wear appropriate personal protective equipment, including gloves and eye protection. Perform all procedures involving PFA in a fume hood and avoid inhalation of vapors.
  5. Remove the PFA and wash the samples at least four times with 1.5 mL of 1× PBS. Gently invert the tube two to three times during each wash.
  6. Transfer the fish to 30% sucrose prepared in PBS.
  7. Incubate the fish in a cold room overnight until the specimen sinks to the bottom of an upright tube (approximately 12 h), indicating adequate sucrose penetration.
  8. Remove excess sucrose solution by gently blotting the fish on a laboratory tissue.
  9. Transfer the fish to storage tubes.
  10. Store the samples at −80°C until imaging.
    NOTE: Process samples within 3 months to minimize fluorescent protein degradation and signal loss.
  11. Transport the fixed fish to the cryostat room on dry ice.
  12. Place the fish flat in a plastic mold with one sagittal side facing downward.
  13. Add Optimal Cutting Temperature (OCT) compound until the fish is fully covered.
  14. Freeze the mold at −20°C or in the cryostat until the OCT turns white.
    NOTE: Position the fish as flat as possible before freezing to facilitate consistent sectioning.
  15. Cryosection the zebrafish in the sagittal plane to remove the skin for subsequent whole-mount imaging.
    1. Cool the cryostat chuck.
    2. Apply room-temperature OCT to the chuck.
    3. Press the OCT fish block firmly into the OCT layer.
    4. Freeze the chuck and block until the newly applied OCT turns white.
    5. Mount the chuck in the cryostat.
    6. Cryosection the block at a thickness of 14 µm until the blade reaches the specimen.
      NOTE: Specimen shifting may occur during freezing, causing the fish to sit higher in the block than expected. Entry into the specimen is indicated by the appearance of translucent tissue replacing the opaque OCT background.
    7. Reduce the section thickness to 7 µm.
    8. Continue sectioning for one additional cut.
      NOTE: This procedure removes the skin while preserving a uniform sagittal profile across the specimen.
    9. Confirm the endpoint by identifying skin in the collected sections.
      ​NOTE: Skin appearance in sections provides a more reliable indicator than eye morphology. Perform this procedure only when the skin obscures imaging of the tumor microenvironment.
  16. Warm the chuck gently with your hands.
  17. Remove the OCT block from the chuck using forceps. Avoid disturbing the cut surface of the specimen.
  18. Trim excess OCT surrounding the fish while the OCT remains frozen.
  19. Remove the fish from the OCT block using forceps once the OCT softens to a viscous consistency.

5. Mounting and Imaging of Fish with Skin Removed

  1. Place the fish in a glass-bottom dish with the sectioned sagittal side facing the glass surface.
    NOTE: Maintain the orientation of the fish during handling. Identifying the sectioned side may become difficult after the fish is removed from the OCT block.
  2. Prepare 1%–1.5% low-melting agarose.
  3. Apply approximately 30–50 µL of agarose to the dorsal and ventral sides of the fish to secure the sample.
    NOTE: Avoid placing agarose beneath the fish because it can reduce image quality. Use only the minimum amount of agarose required for stabilization.
  4. Keep the sample on ice during transport. Image the sample within 2 h of mounting.
  5. Transport the sample to the confocal microscopy facility.
  6. Image the sample using confocal microscopy.
    1. Use a standard laser-scanning confocal microscope with a resonant scanner. A two-photon microscope may be used when autofluorescence limits image quality. Use a corrected 20× objective lens with a numerical aperture of at least 0.80.
    2. Set the pinhole to 1.0 Airy unit (AU) for all imaging channels.
    3. Use a 488 nm laser for EGFP excitation and a 594 nm laser for mCherry excitation. Keep laser output below 25 mW. Adjust detector gain and laser power using a range indicator lookup table (LUT) to avoid pixel saturation and black clipping. Do not exceed a detector gain of 800 V because higher voltages introduce excessive noise.
    4. Configure image acquisition parameters as follows.
      1. Use a voxel size of 0.42 × 0.42 × 6.0 µm (x, y, z) at a resolution of 1024 × 1024 pixels and a Z-step interval of 6.0 µm.
      2. Acquire spectral image stacks through the full volume of interest, typically spanning a total Z-stack acquisition range of 90–120 µm (15–20 optical sections).
      3. Initiate image acquisition in the fluid space immediately above the tissue surface and continue through the full target imaging depth to ensure complete coverage of the specimen.
      4. Acquire images using highly sensitive photodetectors operating in 16-bit acquisition mode.
    5. Acquire images using unidirectional line scanning at a resonant scan frequency of 33.3 kHz (approximately 1.58 µs pixel dwell time) with 8× line averaging.

6. Image Analysis and Quantification

  1. Image visualization using Fiji (version 1.54p).
    1. Open the confocal z-stack images.
    2. Merge colors to see channel overlay by selecting Image > Color > Merge Channels.
    3. Generate a projection of the full z-range containing the imaging region by selecting Image > Stacks > Z Project. Enter the slice range of interest, as well as Max Intensity projection type. This will display the 2D projection of the image.
      NOTE: For larval fish, it is feasible to capture the whole TME by confocal microscopy. Therefore, we recommend the volumetric normalization and quantification method for the TME analysis. For older fish, it is impossible to capture the whole TME by confocal microscopy; therefore, we recommend applying the area-based normalization and quantification method. Based on our analysis and quantification of the larval fish, both methods can provide reliable biological results.
  2. Volumetric normalization and quantification of the TME in the larval fish.
    1. Quantifying tumor volume using MATLAB R2022b (download the package from the Matlab website) and Bio-Formats package (download from the openmicroscopy website).
    2. The user runs a script that performs the following tasks:
      1. Prompt the user to select a .CZI file from their computer.
      2. Read the image and extract the data from the channel containing the tumor region.
      3. Identify the region for each slice that is the tumor region through a threshold of fluorescence (can be through user input or machine learning), keeping the same threshold for all analyzed images for consistency.
      4. Outline the tumor region and display the image with the tumor region outlined in a collage of all slices for user verification.
      5. Calculate the area of the tumor region per slice in pixels and convert to mm2 using the scale of the image and imaging settings.
      6. Multiply the area of the tumor region by the depth (stored in the file’s metadata) to get the volume of each slice.
      7. Sum the calculated values of each slice to obtain the total volume of the imaged tumor and its surrounding region in voxels or mm3.
      8. Scale both volume measurements by multiplying by 104 for tumor volume and 106 for the surrounding region. This will create a more reasonable cell number:volume ratio during normalization.
    3. Quantifying cell number.
      1. Open the confocal z-stack images.
      2. Merge colors to see channel overlay by selecting Image > Color > Merge Channels.
      3. Generate a 3D view of the full z-range containing the imaging region by selecting Image > Stacks > 3D Project. Select Brightest Point as the projection method and other settings to the desired values.
      4. Due to the small number of cells, manually count and tabulate the cells, separating cells located within the tumor boundary from those located outside the tumor boundary.
        NOTE: Cells on the boundary of the tumor region are counted as being outside.
    4. Normalization.
      1. For infiltrating cells inside the tumor region, divide the number of cells by the scaled tumor volume. For the cells outside the tumor region, divide the number of cells outside the tumor volume by the scaled volume of the full image minus the tumor volume.
      2. Generate a table that includes the following headings: “fish identification”, “tumor volume”, “# of cells inside tumor region”, “# of cells outside tumor region”, “normalized cells inside”, “normalized cells outside”.
  3. Area-based normalization and quantification of the TME.
    1. Define the tumor region of interest (ROI) using the Rectangle or Freehand selection tool to outline the tumor.
    2. Add the ROI to the ROI Manager by pressing T or by selecting Analyze > Tools > ROI Manager > Add.
    3. Measure the ROI area using Analyze > Measure.
    4. Confirm that Area is selected under Set Measurements before recording measurements.
    5. Save the ROI in the ROI Manager.
    6. Apply the same ROI to all images using ROI Manager > More > Multi-Measure.
    7. Do not resize the ROI between images.
    8. Manually count cells within the ROI. If cells are more, count cells using the Cell Counter plugin by selecting Plugins > Analyze > Cell Counter.
    9. Mark each counted cell by left-clicking when its centroid falls within the ROI.
    10. Scroll through the z-stack to verify cell identity.
    11. Avoid counting the same cell more than once across multiple image planes.
    12. Count infiltrating cells separately if they contact or lie within the tumor boundary.
    13. Record infiltrating cells as a distinct category even when the cell centroid falls outside the ROI.
    14. Convert the ROI area from pixels to mm2.
    15. Obtain the pixel width, pixel height, and Z-step interval from Image > Properties.
    16. Calculation of normalized cell counts.
      1. Divide the raw cell count for each fish by the corresponding ROI area value.
      2. Record the resulting value as the normalized cell count.
      3. For absolute density measurements, calculate the physical area using the image calibration values obtained from Image > Properties.
      4. Divide the raw cell count by the calculated area (mm2).
      5. Record the result as cell density expressed as cells/mm2.

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Resultados

El flujo general del experimento se muestra en la Figura 1. Se cruzaron peces cebra adultos Tg(cd4-1:mCherry) con peces cebra Tg(dβh:MYCN;dβh:EGFP) para generar descendencia transgénica compuesta en la que las células CD4+ y las células que expresan dβh se marcaron diferencialmente con fluorescencia mCherry y EGFP, respectivamente. Los embriones se seleccionaron según los marcadores fluorescentes adecuados y posteriormente se analizaron mediante imágenes...

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Discusión

El protocolo descrito aquí utiliza el pez cebra como un modelo in vivo para visualizar y cuantificar células inmunitarias dentro del microentorno tumoral (TME). Varios factores son fundamentales para la implementación exitosa de este enfoque. Primero, la selección y validación adecuadas de líneas transgénicas compuestas de pez cebra son esenciales. Las larvas deben examinarse para verificar los patrones esperados de expresión fluorescente y, si es necesario, confirmarse mediante genotipificación. Debe verificars...

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Divulgaciones

Los autores declaran que no tienen intereses en competencia.

Agradecimientos

Agradecemos el apoyo de los National Institutes of Health (NIH; 1UL1TR001430, CA21059 y NS140967), la American Cancer Society (RSG-17-204-01-TBG), la National Science Foundation (1911253) y la Alex’s Lemonade Stand Foundation para H.F.; así como de una beca Warren Alpert Distinguished Scholars para X.Q. Y.W. recibió apoyo del Programa de Oportunidades de Investigación para Pregrado de la Universidad de Boston (UROP) y del Programa de Becas de Verano de St. Baldrick’s. M.V. recibió apoyo del Programa de Becas de Verano de St. Baldrick’s. El contenido de este artículo es únicamente responsabilidad de los autores y no representa necesariamente las opiniones oficiales de los NIH.

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Materiales

Lista de materiales utilizados en este artículo
NombreEmpresaNúmero de catálogoComentarios
200 µL de punta de carga para gelesMilliporeSigma1022-0600Utilizado para la manipulación de larvas
Plantas artificialesThe Hidden Reef62874200000000000Enriquecimiento opcional para reproducción
LejíaFisher Scientific50371500Utilizada para la descontaminación de embriones
Complemento para contador de célulasNational Institutes of Health (NIH)N/AComplemento de Fiji utilizado para la cuantificación celular
Computadora y monitorHP2TB68A8#ABAUtilizado para la adquisición y análisis de imágenes
Microscopio confocalZeissLSM 710Utilizado para imágenes de larvas y adultos
Molde criogénico, plásticoEpredia22-050-160Utilizado para inclusión en OCT
CriostatoThermo Fisher Scientific95-664-0EC70Utilizado para corte criogénico
Hielo secoFisher ScientificN/AUtilizado para el transporte de muestras
Software FijiNational Institutes of Health (NIH)Version 1.54pSoftware para análisis de imágenes
Red de malla finaCarolina Biological651345Utilizada para la recolección de embriones
Incubadora para pecesVWR35960-056Mantenida a 28.5°C
PinzasFisher Scientific10-316BUtilizadas para el manejo de muestras
Placa de fondo de vidrioIBIDI81218-200Utilizada para imágenes
Agarosa de bajo punto de fusiónProlab Supply9012-36-6Utilizada para montaje de larvas y adultos
Tanque de apareamiento (tanque principal, divisor, sección con rejilla)Carolina Biological161937Utilizado para la reproducción de pez cebra
Agitador rotatorioMidsciR2D-30Utilizado para fijación durante incubación nocturna
Compuesto OCTTissue-Tek4583Medio de inclusión para corte criogénico
Paraformaldehído (PFA)Sigma Aldrich158127-500GFijador
Placa de Petri, plásticaMilliporeSigmaBR452000Utilizada para el manejo de embriones y larvas
Solución salina tamponada con fosfato (PBS)Gibco14080-055Utilizada para lavados y procedimientos de fijación
Pipeta de transferencia plásticaFisher Scientific50-998-100Utilizada para la transferencia de embriones y larvas
Cuchilla de afeitarFisher Scientific12-640Utilizada durante la preparación de muestras
Medidor de conductividadYSIEC300ACC-04Utilizado para verificar la salinidad del agua de rotíferos
RotíferosThe Hidden Reef116151603Utilizados para el cultivo de larvas
Tiosulfato de sodioThermo Fisher ScientificAAA1762936Utilizado para la neutralización de lejía
SacarosaFisher ScientificS25590AReactivo de crioprotección
Tricaina metanosulfonatoWestern ChemicalNC0872873Anestésico; vendido por Fisher Scientific
TenazasFisher Scientific12-000-122Utilizadas para el manejo de muestras
Microscopio de fluorescencia invertidoLeicaM165Utilizado para cribado de fluorescencia

Referencias

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