Description
Overview
FireGene Trachea Dissociation Kit is a specialized enzymatic solution for isolating high-quality single-cell suspensions from tracheal tissue. Designed to support respiratory research and single-cell applications, this kit offers exceptional cell recovery and viability, ideal for understanding airway biology and disease mechanisms.
Background Information
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Driven by Clinical and Scientific Research Needs:
- Studying respiratory diseases like tracheitis, tracheal tumors, and airway inflammation requires understanding cell-level heterogeneity.
- Traditional dissociation approaches often compromise rare or delicate cell types.
- This kit enables:
- Identification of epithelial cells, ciliated cells, immune cells, and more from tracheal samples.
- Discovery of biomarkers and pathways related to inflammation and remodeling.
- Support for targeted therapy development and precision medicine in respiratory health.
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Background of Technological Development:
- Mechanical and chemical methods lack consistency and damage viable cells.
- FireGene’s approach integrates:
- Optimized enzymatic digestion tailored to tracheal matrix structure.
- Adjusted protocols for reaction time, temperature, and reagent ratios.
- Superior cell viability, efficiency, and reproducibility in tracheal tissue dissociation workflows.
Detection Principle
- Employs a multi-enzyme digestion strategy:
- Trachea tissue is minced into small fragments.
- A controlled sequence of enzyme application is used.
- Enzymes gradually break down extracellular matrix and cell-cell junctions.
- Result:
- A clean, viable single-cell suspension optimized for scRNA-seq, immune profiling, and respiratory disease modeling.
Specifications
| Applications | Single-cell sequencing, cell culture or other cell-related detections |
| Compatible Sample Types | Trachea tissue |
| Supported Instruments | Water bath, horizontal centrifuge, cell counter |
| Storage | -20 °C |
| Shelf-life | 24 months |
Kit Components
10 Reactions
| Component | 10 Reactions/Kit |
| Trachea DS 1 (Trachea Dissociation Solution) | 1*30 mL |
| Trachea DS 2 (Trachea Dissociation Solution) | 1*30 mL |
50 Reactions
| Component | 50 Reactions/Kit |
| Trachea DS 1 (Trachea Dissociation Solution) | 5*30 mL |
| Trachea DS 2 (Trachea Dissociation Solution) |
5*30 mL |
Product FAQ
1. Q: When dissociating tracheal tissue, according to the procedure, first digest with Dissociation Buffer 1 for 15-30 minutes. After centrifugation, it is found that the tissue blocks are still large and cannot be fully resuspended with Dissociation Buffer 2. What is the reason, and how to handle the insufficiently digested tissue blocks?
A: The large size of tissue blocks is due to the failure of Dissociation Buffer 1 to fully degrade the cartilage and fibrous tissue in the outer layer of the trachea. Handling methods: ① Re-mince the centrifuged tissue blocks to less than 0.2mm³ (half the size of the first mincing), rinse repeatedly with 1mL PBS to remove residual Dissociation Buffer 1; ② Add 2mL of Dissociation Buffer 1 supplementally, incubate at 37℃ for 10 minutes, and shake gently every 3 minutes to enhance enzymolysis effect; ③ After centrifugation again, the tissue blocks can be reduced to an appropriate size and evenly dispersed when resuspended with Dissociation Buffer 2, ensuring sufficient subsequent enzymolysis.
2. Q: When dissociating tracheal tissue from asthmatic model mice, a large amount of viscous secretions appear in the cell suspension, which wrap the cells and cause filtration difficulties. How to remove these secretions?
A: The viscous secretions are formed by the mixture of mucin secreted by asthmatic tracheal tissue and enzymolysis products. Removal methods: ① Add 100μL of 0.1% hyaluronidase (self-prepared, non-toxic to cells) during digestion with Dissociation Buffer 2 to degrade the mucopolysaccharide components in the secretions; ② Centrifuge the cell suspension at 4℃, 500×g for 3 minutes before filtration, and discard the upper viscous layer; ③ Rinse the 70μm cell sieve 3 times with PBS containing 2% FBS to ensure the secretions flow out with the filtrate and avoid sieve clogging, which can increase the filtration speed by 50%.
3. Q: When using a hybridization oven for enzymolysis of Dissociation Buffer 2 at a rotation speed of 20-30 rpm, it is found that the liquid in the centrifuge tube forms a vortex, causing tissue blocks to aggregate at the bottom of the tube and fail to be enzymolyzed. What is the reason, and how to avoid vortex formation?
A: The vortex is formed because the tracheal tissue contains high-density cartilage debris, which drives the liquid to rotate when the rotation speed of the hybridization oven is too high. Avoidance methods: ① Reduce the rotation speed of the hybridization oven to 15-20 rpm to reduce the liquid rotation force; ② Put 1 sterile glass bead (2mm in diameter, self-prepared) into the centrifuge tube, and use the collision of the glass bead to disperse the tissue blocks; ③ Manually invert the centrifuge tube once every 20 minutes to ensure full contact between the tissue blocks and Dissociation Buffer 2, which can improve the enzymolysis uniformity by 70%.
4. Q: After opening Dissociation Buffer 1 and Dissociation Buffer 2 in the kit, they are stored at -20℃ for 5 months. When used, it is found that the enzymolysis efficiency of Dissociation Buffer 2 decreases (the cell yield is only 55% of the original). Is the reagent invalid, and how to judge and remedy it?
A: The validity period of the opened dissociation buffers stored at -20℃ is 3 months (2 years for unopened ones), and 5 months of storage may lead to decreased enzyme activity. Judgment methods: ① Mix 50μL of Dissociation Buffer 2 with a small amount of minced tracheal tissue, incubate at 37℃ for 30 minutes. If there is no obvious change in the tissue blocks, it indicates insufficient enzyme activity; ② Remedial methods: Increase the dosage of Dissociation Buffer 2 by 50%, extend the digestion time to 2 hours, and add 50μL of 1% BSA (self-prepared) to Dissociation Buffer 2 at the same time to enhance enzyme stability. This can restore the cell yield to about 80% of the original (not recommended for single-cell sequencing).
5. Q: In Step 10, rinse the digestion tube with 6mL of RPMI 1640 medium. If a large number of epithelial cells (target tracheal cells) are still attached to the tube wall after rinsing, resulting in low cell yield, how to optimize the rinsing operation?
A: The adhesion of epithelial cells is due to the adhesion molecules contained in tracheal epithelial cells, which are easy to attach to the inner wall of the centrifuge tube. Optimization methods: ① Rinse the inner wall of the centrifuge tube with PBS containing 5% FBS before rinsing to form a protective film and reduce cell adhesion; ② Divide 6mL of medium into 3 times for rinsing (2mL each time), and invert the centrifuge tube repeatedly 10 times each time to ensure every area of the tube wall is rinsed; ③ Gently scrape the tube wall with a 1mL pipette tip after rinsing, and then rinse with a small amount of medium, which can increase the recovery rate of epithelial cells by 40%.
6. Q: When dissociating tracheal tissue from newborn mice (within 5 days of birth), the cell viability is only 30%, which is much lower than 75% of adult mice. What is the reason, and how to adjust the operation to protect immature tracheal cells?
A: The cell membrane of immature tracheal cells is fragile and easily damaged by enzymolysis solution and mechanical force. Adjustment methods: ① Shorten the digestion time of Dissociation Buffer 1 from 15-30 minutes to 10-15 minutes, and the digestion time of Dissociation Buffer 2 to 30-40 minutes to reduce enzyme damage; ② Use a water bath for enzymolysis, and shake gently every 5 minutes (avoid violent oscillation) to reduce mechanical impact; ③ In the washing step, replace the conventional PBS containing 5% FBS with PBS containing 10% FBS to enhance cell membrane protection, which can increase the viability of immature tracheal cells to more than 65%.
7. Q: After centrifugation in Steps 5 and 6, it is required to "do not aspirate the tissue". If a small amount of tissue blocks are accidentally aspirated, what impact will it have on the subsequent experiment, and how to recover the aspirated tissue blocks?
A: Aspirating tissue blocks will lead to the loss of target cells, reducing the cell yield by 20%-30%. Recovery methods: ① If the tissue blocks are still in the pipette tip, immediately squeeze the liquid in the tip back into the centrifuge tube; ② If the supernatant has been aspirated, collect the supernatant into a new centrifuge tube, centrifuge at 4℃, 300×g for 8 minutes, discard the supernatant, and combine the precipitate with the original tissue blocks; ③ Add 0.5mL of Dissociation Buffer 2 supplementally during subsequent enzymolysis, and extend the digestion time by 10 minutes to make up for cell loss, which can restore the cell yield to 85% of the normal level.
8. Q: A large number of transparent cartilage debris appear in the cell suspension after dissociation, interfering with cell counting. How to remove these cartilage debris?
A: Cartilage debris is a product of incomplete enzymolysis of the cartilage tissue in the outer layer of the trachea. Removal methods: ① Before filtration after enzymolysis, let the cell suspension stand for 5 minutes, the cartilage debris will settle to the bottom of the tube, and aspirate the upper cell suspension for filtration first; ② When filtering with a 70μm cell sieve, rinse the sieve repeatedly with PBS containing 2% FBS, the cartilage debris will remain on the sieve, and gently scrape it off with tweezers; ③ If there is still residue, perform secondary filtration with a 40μm cell sieve (only retain the filtrate), which can remove more than 90% of the cartilage debris without affecting the recovery rate of epithelial cells.
9. Q: When using FG-BA3311 Red Blood Cell Lysis Buffer to remove red blood cells in Step 15, it is found that the viability of tracheal epithelial cells drops from 80% to 50%. How to optimize the red blood cell lysis operation to reduce damage to epithelial cells?
A: The damage to epithelial cells is due to the slight toxicity of the lysis buffer to tracheal epithelial cells. Optimization methods: ① Dilute the lysis buffer 1-fold with PBS to reduce its concentration; ② Control the incubation temperature at 4℃, shorten the time to 3-4 minutes, and terminate immediately when red blood cell lysis is observed under a microscope; ③ Wash 3 times with PBS containing 10% FBS after lysis (1 time for conventional operation) to fully remove residual lysis buffer, which can maintain the viability of tracheal epithelial cells above 70%.
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