FireGene Gastrointestinal Dissociation Kit - Gut Cell Isolation

Overview

FireGene Gastrointestinal Dissociation Kit is a high-performance enzymatic solution designed to isolate viable single-cell suspensions from GI tissues. Optimized for applications like single-cell sequencing, immune profiling, and gut microbiota research, this kit delivers exceptional results for complex and fragile gastrointestinal samples.

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Background Information

• Driven by Clinical and Scientific Research Needs:

  • Single-cell sequencing of GI tissue is critical for understanding diseases such as IBD (inflammatory bowel disease), colorectal cancer, and intestinal fibrosis.
  • Conventional methods do not fully preserve the diversity of epithelial, stromal, and immune cells.
  • This kit enables:
    • Detailed characterization of cell subsets involved in inflammation, cancer, and tissue repair.
    • Identification of biomarkers and therapeutic targets for precision medicine.
    • Support for regenerative and microbiome-associated GI research.

• Background of Technological Development:

  • Traditional techniques often result in low cell viability and inefficient dissociation.
  • FireGene resolves these limitations by:
    • Using a targeted enzymatic system tailored for gut ECM and cell junctions.
    • Optimizing reaction conditions for reproducibility across different GI tissue types.
    • Delivering high cell recovery rates suitable for sensitive downstream applications.

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Detection Principle

• Uses a multi-step enzymatic digestion approach:
  • GI tissue is cut into small fragments for uniform reagent exposure.
  • Enzymes are applied sequentially, breaking down extracellular matrix and cellular adhesions.
  • The process occurs under carefully regulated incubation conditions.
• Outcome:
  • A high-quality, viable single-cell suspension ideal for scRNA-seq, flow cytometry, and functional GI assays.

Specifications

Applications	Single-cell sequencing, cell culture or other cell-related detections
Compatible Sample Types	Gastrointestinal tissue
Supported Instruments	Water bath, horizontal centrifuge, cell counter
Storage	-20 °C / 4 °C
Shelf-life	24 months at -20 °C 12 months at 4 °C

Kit Components

10 Reactions/kit

Component	Quantity	Storage	Shelf-life
Buffer A	40 mL	-20 °C	24 months
Enzyme B	1 mL	-20 °C	24 months
Enzyme C	100 μL	4 °C	12 months

50 Reactions/kit

Component	Quantity	Storage	Shelf-life
Buffer A	5*40 mL	-20 °C	24 months
Enzyme B	5*1 mL	-20 °C	24 months
Enzyme C	5*100 μL	4 °C	12 months

 

Product FAQ

1.    Q: Is this kit only suitable for mammalian gastrointestinal tissue? Are there differences in dissociation effects on tissues from different parts of the gastrointestinal tract (e.g., gastric mucosa, small intestinal villi, colonic mucosa)? Can it be used for non-mammalian gastrointestinal tissue (e.g., fish intestines)?

A: The kit is suitable for full-segment gastrointestinal tissue (stomach, small intestine, colon, etc.) of mammals such as humans and mice. There are slight differences in dissociation effects on tissues from different parts: small intestinal villi tissue is relatively fragile, so the digestion time is recommended to be shortened to 20-30 minutes to avoid villus structure breakage; gastric mucosa and colonic mucosa contain more collagen fibers, so the digestion time can be extended to 30-40 minutes, and dynamic adjustment is required through quality inspection. It is not suitable for non-mammalian gastrointestinal tissue. The cell membrane structure and fiber components of non-mammalian gastrointestinal cells (such as fish intestines) are significantly different from those of mammals. The enzyme system of the kit cannot accurately degrade their extracellular matrix, which easily leads to incomplete dissociation or a sharp drop in cell viability.

2.    Q: The kit is labeled "10 rxns". Each experiment requires 4mL of Buffer A, 100μL of Enzyme B, and 10μL of Enzyme C. If only 100mg of gastrointestinal tissue (1/2 of the standard dosage) is processed in a single experiment, can the reagent dosage be reduced proportionally?

A: Proportional reduction of reagent dosage is not recommended. Buffer A is not only a reaction medium but also needs to maintain the tissue suspension environment; a volume of 4mL can ensure the full dispersion of tissue fragments. The ratio of Enzyme B to Enzyme C is precisely formulated; reducing the dosage will lead to insufficient enzyme concentration, which cannot effectively decompose the fibers of gastrointestinal tissue and instead increases the cell clumping rate. When processing 100mg of tissue, reagents should still be added according to the standard dosage. During subsequent centrifugation to collect cells, the centrifugation time can be extended (from 5 minutes to 8 minutes) to ensure sufficient cell precipitation and avoid cell loss due to the relatively large liquid volume.

3.    Q: During digestion in Step 2, a water bath requires "manual shaking every 3-5 minutes", and a hybridization oven requires "a rotation speed of 20-30 rpm". What are the core differences in operation effects between the two devices? Which one is more suitable for gastrointestinal tissue dissociation?

A: The core difference lies in "the uniformity of enzyme-tissue contact": the automatic rotation speed of the hybridization oven can keep gastrointestinal tissue fragments suspended in the dissociation solution at all times, avoiding local over-enzymolysis (e.g., tissue at the bottom of the tube) or insufficient enzymolysis (e.g., tissue at the liquid surface) caused by gravity sedimentation. If the manual shaking frequency of the water bath is inconsistent, it is easy to cause over-digestion of small intestinal villi tissue and incomplete dissociation of colonic mucosa tissue. In terms of effect, the hybridization oven is more suitable for gastrointestinal tissue dissociation, especially for fiber-rich tissues such as the colon, with a single-cell yield 18%-25% higher than that of the water bath. If a water bath is used, it is necessary to strictly shake at 3-minute intervals for 10 seconds each time to ensure no tissue settles at the bottom.

4.    Q: Both Step 5 and Step 6 require filtration with a 70μm cell sieve. If the laboratory lacks a 70μm cell sieve, can it be replaced with a 50μm or 100μm cell sieve? What impact will the replacement have?

A: A 50μm or 100μm cell sieve cannot be used as a replacement. The pore size of the 50μm sieve is too small and will retain normal single cells in gastrointestinal tissue (e.g., the diameter of small intestinal epithelial cells is about 12-18μm; although they can pass through, the sieve is easily blocked by fiber fragments, leading to filtration difficulties and even cell extrusion to reduce viability). The pore size of the 100μm sieve is too large and cannot effectively filter undigested tissue fragments (e.g., gastric mucosa fiber fragments). These fragments will enter subsequent steps along with cells, interfering with the accuracy of cell counting and may even block the single-cell sequencing chip. If a 70μm cell sieve is lacking, it is necessary to contact the supplier for emergency deployment or select a compatible 70μm cell sieve of the same brand (product number: BS70-C); random replacement is not allowed.

5.    Q: The instruction manual mentions that "DMEM medium can replace RPMI 1640 medium". After replacement, is it necessary to adjust centrifugation parameters or digestion time? Is there any difference in the impact of the two media on the viability of gastrointestinal cells?

A: There is no need to adjust centrifugation parameters (still 4℃, 300×g for 5 minutes) or digestion time after replacement. Both DMEM and RPMI 1640 are common basal media for mammalian cells. Although they differ in glucose and amino acid content, both can provide a suitable osmotic pressure (280-320mOsm/kg) and pH (7.2-7.4) for gastrointestinal tissue dissociation, with no impact on enzymolysis efficiency. The impact on the viability of gastrointestinal cells is minimal: experimental data shows that the difference in cell viability after dissociating mouse small intestinal tissue with the two media is ≤6%. The medium can be selected based on the existing inventory in the laboratory without deliberate replacement.

6.    Q: Step 7 uses PBS containing 5% FBS to wash cells. If serum-free operation is required for the experiment (e.g., some cell sorting experiments), can PBS without FBS be used as a replacement? Or can serum substitutes (e.g., BSA) replace FBS?

A: PBS without FBS cannot be used as a replacement. The core function of FBS is to neutralize residual enzyme activity (Enzyme B and Enzyme C) to prevent enzymes from continuing to damage cells; PBS without FBS cannot neutralize enzyme activity, which will lead to a more than 30% drop in cell viability after washing. If serum-free operation is required, PBS containing 1% BSA can be used to replace PBS containing 5% FBS. BSA can play a similar enzyme-neutralizing role and does not contain serum components; however, it should be noted that after replacing with BSA, the washing time needs to be extended from 5 minutes to 8 minutes to ensure complete neutralization of enzyme activity and avoid residues affecting subsequent experiments.

7.    Q: Enzyme C needs to be stored at 4℃ in the dark. If it is left at room temperature for 30 minutes (e.g., forgotten to be put back in the refrigerator after use), can it still be used? What consequences will occur if Enzyme C is stored at -20℃ for a long time?

A: It can still be used after being left at room temperature for 30 minutes, but it must be immediately put back in the 4℃ refrigerator and fully mixed before use. Enzyme C is an active enzyme preparation; short-term storage at room temperature (≤30 minutes) results in ≤10% activity loss, which does not affect the dissociation effect. Long-term storage of Enzyme C at -20℃ is not allowed. Low temperature will cause Enzyme C to denature and inactivate (after 24 hours of freezing at -20℃, activity decreases by more than 70%), making it unable to work synergistically with Enzyme B to decompose gastrointestinal tissue fibers, leading to dissociation failure. The correct storage condition for Enzyme C is 4℃ in the dark, with a validity period of 1 year, which must be strictly followed.

8.    Q: If there are many red blood cells in the cell suspension after dissociation and FG-BA3311 Red Blood Cell Lysis Buffer is needed to remove them, after which step should this operation be performed? What should be noted during lysis to avoid damaging gastrointestinal cells?

A: This operation should be performed after Step 7 and before Step 8. Specific operation: After discarding the supernatant in Step 7, add 1mL of FG-BA3311 Red Blood Cell Lysis Buffer, incubate on ice for 5 minutes (to avoid damaging gastrointestinal cells due to room-temperature incubation), centrifuge at 4℃, 300×g for 5 minutes, discard the supernatant, then resuspend with 5mL of PBS containing 5% FBS (add an additional washing step), and then proceed to Step 8. Precautions: ① The lysis time should not exceed 8 minutes; ice incubation can reduce the toxicity of the lysis buffer to gastrointestinal cells; ② If there are too many red blood cells (e.g., gastric mucosa tissue contains a large number of capillaries), lysis can be repeated once, but an additional PBS washing step is required to avoid residual lysis buffer affecting subsequent experiments (e.g., interference with fluorescence signals in cell sorting).