Apr 29, 2026
Production of Clarified Tissue Extract (CTE)
- Christina M. Ferreira1,
- Kellie P. Burris1,
- Rebecca L. Bell1,
- Jie Zheng1
- 1U.S. Food and Drug Administration, College Park, MD
Protocol Citation: Christina M. Ferreira, Kellie P. Burris, Rebecca L. Bell, Jie Zheng 2026. Production of Clarified Tissue Extract (CTE). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn5o86g5d/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: April 06, 2026
Last Modified: April 29, 2026
Protocol Integer ID: 314551
Keywords: Tomato Intracellular Fluid, biochemical analysis, sterilization, homogenization, filtration, production of clarified tissue extract, clarified tissue extract, vegetable tissue extract via freeze, using ripe tomato tissue, clarified fruit, vegetable tissue extract, ripe tomato tissue, resulting extract, assisted tissue disruption, tissue disruption, preparation, cte, soluble component
Disclaimer
This protocol was refined using AI.
Abstract
This protocol describes the preparation of a clarified fruit or vegetable tissue extract via freeze–thaw–assisted tissue disruption, mechanical homogenization, and sequential filtration and centrifugation. The resulting extract contains soluble components derived from disrupted cellular compartments and is suitable for downstream microbiological, biochemical, or analytical applications. This protocol was developed and validated using ripe tomato tissue; application to other plant matrices or developmental stages may require parameter optimization.
Materials
NOTE: All items that can be sterilized should be sterilized.
Red-ripe tomatoes (Fresh, intact, harvested at 117-151 days post pollination)
For Surface Sterilization and Sample Prep:
Aluminum foil - Large Sheets
70% Ethanol
Sterile deionized water
Lab gloves
Sharp knife that can withstand being flame sterilized
Cutting board
Kimwipes or papertowels
Large Aluminum Disposable Pans
For Homogenization:
Blender base(For homogenization)
Blender carafes (For homogenzation)
Sterile Scoop or large spoon
For Filtration:
Large Buchner Filter funnel (200mm)
Cheese cloth (Grade 90)
Side arm flask (4-liter capacity)
Side arm flask (2-liter capacity)
Conical tubes (50 mL capacity)
Parafilm Tape
vacuum pump or vacuum line
100ml syringe
Glass screw cap bottles (500 mL capacity)
Centrifuge capable of 25,500 xg speed for 50ml conical tubes
Glass powder funnel (ex. Corning 6220-150)
Fiber glass wool (Sigma-aldrich CLS3950); torn into portions (see below) using caution
Troubleshooting
Problem
Contamination of samples
Solution
Ensure all surfaces and equipment are sterile; use aseptic techniques throughout.
Problem
Low yield of CTE
Solution
Check the efficiency of the blending and filtration steps; ensure proper centrifugation speed and time.
Safety warnings
Handle reagents, especially ethanol, with care to minimize fire risk. Wear appropriate PPE, including gloves and eye protection.
Use caution when handling sharp instruments to prevent cuts. Work on a stable surface, keep fingers clear of the blade, and cut away from your body. Use only sharp, undamaged tools and wear additional PPE (e.g., cut-resistant gloves) if required.
Store or dispose of sharps immediately after use and never leave them unattended. Follow institutional first aid and reporting procedures in case of injury.
‱ WARNING: Fiberglass Wool Handling
Fiberglass wool is an irritant that can cause skin, eye, and respiratory irritation upon contact or inhalation.
Required Safety Measures:
- Handle only in a fume hood when cutting or portioning into segments to prevent airborne fibers.
- Wear appropriate PPE: lab coat, gloves, and safety goggles.
- Use a particulate respirator (e.g., N95 or higher) if there is any risk of airborne fibers.
- Avoid direct skin contact; do not rub exposed areas.
- Wash hands thoroughly after handling.
Before start
Ensure all equipment, reagents, tools and supplies that come into contact with fruit prior to final product are sterile, and maintain sterile technique throughout to prevent microbial contamination.
The final product represents a clarified tissue extract, not intact intracellular fluid or natural exudate.
Fruit Selection and Preparation
Harvest or purchase tomatoes at the preferred stage of development.
Transport samples on ice packs to the laboratory.
Inspect fruit upon arrival, and discard any with visible damage.
Surface Steriliation
Place tomatoes on sterile aluminum foil.
Preparation of whole tomatoes arranged on aluminum foil within a laminar flow biosafety cabinet. Individual tomatoes are spaced apart on foil sheets to maintain sterility and prevent cross-contamination during experimental handling. A sterile water container is positioned to the right for rinsing or treatment steps.
Apply 70% ethanol, by spraying, directly to the surface and agitate, by rubbing, manually with gloved hands that have been cleaned with 70% ethanol every 2–3 minutes for 15 minutes.
Rinse by immersing twice in sterile deionized water, using a fresh bath each time.
Tissue Processing and Storage
Aseptically cut tomatoes into ~1-inch segments; peeling is not required unless specified by the research question.
Aseptic processing of tomatoes on a laboratory bench. Personnel wearing lab coats and gloves dice whole tomatoes on a sanitized cutting board using flame sterilized knives. The chopped material is transferred into an sterile aluminum tray (which will be covered with sterile aluminum foil) for collection prior to downstream processing.
Transfer segments to a sterile pan and cover with sterile foil.
Store at −80 °C for ≥48 hours to promote cellular disruption.
NOTE: The freeze–thaw step is critical for efficient cellular disruption and release of soluble intracellular and apoplastic contents.
Thawing and Homogenization
Transfer frozen samples to 30 °C and thaw overnight to ambient temperature.
Using a sterile scoop/spoon, Homogenize thawed tissue, in batches, in a sterile blender at high speed for 30 seconds to generate a uniform slurry.
Batch size should be determined by the size of your blender carafe's fill line.
Homogenization of thawed tomato tissue using a laboratory blender. Thawed, chopped tomato samples are transferred in batches, using a sterile scoop/spoon, into a sterile blender and processed at high speed for 30 seconds to produce a uniform slurry. Samples are kept covered with foil prior to homogenization to minimize contamination.
Gross Filtration
Place the large Buchner filter funnel over a 4L side-arm flask, and use parafilm to seal the connection points between the two. Line the funnel with four layers of sterile Grade 90 cheesecloth.
Filtration setup for homogenized tomato slurry. The blended sample is poured through layered sterile gauze secured over an Erlenmeyer flask to remove large particulates. Filtrate is collected in the flask for subsequent analysis.
Apply vacuum to a 4 L side-arm flask.
Filter the homogenized slurry and collect the filtrate.
Filtration of homogenized tomato slurry through sterile gauze. The slurry is passed through layered gauze positioned over an Erlenmeyer flask to remove coarse debris, allowing filtrate to collect in the flask below for downstream processing.
Clarification by Centrifugation
Aliquot filtrate into 50 mL conical tubes.
Filtered tomato homogenate prior to centrifugation. The filtrate is aliquoted into a conical centrifuge tube and prepared for subsequent centrifugation to separate remaining particulates.
Centrifuge at 25,500 × g for 30 minutes.
Collect supernatants and pool.
Separation of tomato homogenate following centrifugation. Centrifugation results in supernatant and a compact pellet of particulate material at the bottom of the conical tube, enabling collection of the supernatant for downstream applications.
Fine Filtration
Remove the plunger from a 100 mL syringe.
Tightly pack the syringe barrel with glass fiber wool until full.
100 mL syringe densely packed with sterile fiberglass wool.
Place the glass powder funnel onto a 1 L side-arm flask. Seal the funnel–flask junction with Parafilm, as in the previous filtration setup.
Vacuum filtration setup with glass powder funnel seated on a side-arm Erlenmeyer flask, sealed with Parafilm and connected to vacuum line for sterile filtration.
Place the glass fiber packed syringe onto the top of the powder funnel. Apply vacuum to secure the syringe in place.
Custom filtration assembly with a 100 mL syringe packed with sterile fiberglass wool mounted in a glass funnel on a vacuum flask for vacuum-assisted filtration.
Pour the pooled supernatant into the syringe for vacuum filtration. Be sure to continue adding glass fiber wool as needed to keep the syringe fully packed during filtration.
Collect and pool the clarified filtrate.
Storage and Expected Outcome
The pooled clarified filtrate now represents a particle-reduced tomato tissue extract containing soluble metabolites, ions, and macromolecules derived from disrupted fruit tissue.
Aliquot clarified extract into sterile, appropriately sized containers as needed to minimize repeated freeze–thaw cycles.
Store aliquoted extract at −20 °C or below until use.
Post-Storage Handling
Remove stored extract and thaw at 4 °C overnight.
Following thawing, allow the sample to remain undisturbed to preserve any stratified or dissociated material.
If required for downstream applications, carefully recover the desired fraction without mixing; additional clarification (e.g., low-speed centrifugation or filtration) may be performed if particulates are present.
Do not use samples that have been thawed for longer than 48 hours.