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Cat. No.: LTSCDE-50 (for 50T)

Cat. No.: LTSCDE-100 (for 100T)

Description

The Ligament Tissue Single-Cell Dissociation Enzyme is specifically designed for preparing single-cell suspensions from ligament tissues of humans, mice, rats, and other animals. Through the gentle enzymatic action of a multi-component enzyme blend, ligament tissues are efficiently dissociated into single-cell suspensions. This process achieves both high yield and high cell viability, making the product suitable for downstream applications such as single-cell sequencing, flow cytometry analysis, flow cytometry sorting, primary cell culture, and 3D organoid culture.

Components

• Bottle A: Enzyme Powder
• Bottle B: Enzyme Dissolution Solution

Features

• Ready-to-use, one-step format Simply dissolve the provided enzyme powder in the supplied solution to begin experiments. Unlike imported competitors that require dissolving three to four enzymes separately, calculating volumes, and mixing them together, this product eliminates weighing, calculations, and multi-step operations—reducing error risk.
• Multi-component enzyme blend Developed and manufactured in-house, the enzyme contains more than eight components tailored to different tissue matrices. This enables more thorough digestion compared to imported collagenase IV, DNase, or competitor blends of three to four enzymes. It offers shorter dissociation times, higher yields, stronger cell viability, intact antigen epitopes, and lower production costs.
• Short dissociation time With over eight proprietary tissue-specific enzymes, most embryoid tissues can be fully dissociated within 10–15 minutes. In contrast, collagenase IV, DNase, or competitor blends often require 1–3 hours, or even 2–3 hours for complete dissociation.
• High cell yield The multi-enzyme system ensures complete digestion of extracellular matrices, releasing single cells thoroughly with minimal tissue clumps, resulting in higher yields.
• Strong cell viability Gentle dissociation enzymes specific to different tissues avoid the damage caused by trypsin. Shorter digestion times minimize ex vivo stress and prevent over-digestion, yielding highly viable single cells with intact antigen epitopes.
• Intact antigen epitopes Higher enzymatic efficiency and shorter digestion times minimize tissue damage, preserve cell integrity, and maintain strong viability. This ensures antigen epitopes remain intact, facilitating antibody staining and making dissociated cells ideal for flow cytometry analysis and sorting.

Protocol

1. Dissolving Enzyme Powder

Perform this step when using the kit for the first time.

• Tap the enzyme powder in Bottle A firmly to the bottom.

• Use a pipette to aspirate the dissolution buffer from Bottle B, repeatedly pipette to suspend the enzyme powder in Bottle A, and then transfer the mixture into Bottle B.

• If necessary, cut the pipette tip slightly wider with scissors to ensure all enzyme powder is transferred from Bottle A to Bottle B.

• Tighten the cap of Bottle B, invert repeatedly until the enzyme powder is fully dissolved. If needed, place Bottle B on a shaker until complete dissolution.

• The fully dissolved enzyme solution should appear clear with a light brown tint.

2. Filtration and Aliquoting of Dissociation Enzyme

After the enzyme powder in Bottle A is completely dissolved in the dissolution buffer of Bottle B:

• If sterile tissue dissociation is required, filter the enzyme solution using a syringe and a 0.22 μm sterile filter, then aliquot.

• Recommended aliquot: 5 mL per vial, dispensed into Bottle A.

• Store aliquots immediately at –20 °C or –80 °C, stable for up to 6 months.

• If not fully used in one experiment, aliquots can be refrozen for later use, but avoid more than three freeze–thaw cycles. Each cycle reduces enzyme activity; compensate by increasing enzyme volume to maintain dissociation efficiency.

3. Enzymatic Dissociation Procedure

(1) Preparation

• Prepare crushed ice.

• Thaw single-cell dissociation enzyme and stop solution on ice.

• Preheat tissue dissociation instrument.

• Pre-cool PBS.

• Prepare sterile surgical instruments (ophthalmic scissors, forceps), 40 μm sterile cell strainer, centrifuge tubes, and pre-cool centrifuge.

(2) Tissue Collection

• Collect tissue under sterile conditions.

• Immediately place in cold PBS.

• Process within 4 hours if possible. For longer storage, use High-Activity Tissue Preservation Solution.

(3) Tissue Washing

• Wash tissue with cold PBS 1–2 times to remove blood and necrotic debris.

• Record tissue weight.

(4) Tissue Mincing

• Transfer washed tissue into a dissociation tube.

• Mince into 1–2 mm³ pieces using sharp ophthalmic scissors until paste-like.

• The finer the mince, the more efficient the enzymatic digestion and the shorter the dissociation time.

• If using a tissue dissociation instrument, strict mincing is not required; good dissociation results can still be achieved.

(5) Tissue Digestion

• Add appropriate volume of single-cell dissociation enzyme to the minced tissue in the dissociation tube.

• Invert tube to mix thoroughly.

① With tissue dissociation instrument:

• Operate according to instrument guidelines.

• This product is compatible with all domestic and imported brands.

• Note: Dissociation efficiency is higher than self-prepared or competitor enzymes; digestion time can be reduced by at least half.

• Check dissociation status every 5–10 minutes.

• Complete digestion time varies by tissue (5–50 minutes).

• Terminate digestion once complete to avoid over-digestion.

② Without instrument (manual dissociation):

• Strictly mince tissue as described.

• Depending on tissue and enzyme volume, use 1.5 mL or 5 mL EP tubes.

• Mix enzyme thoroughly with tissue.

• Cut the tip of a 1 mL pipette slightly wider with scissors.

• Pipette mixture vigorously; if clogging occurs, mince tissue further or widen tip.

• Pipette mixture repeatedly (≥10 times), then incubate at 37 °C in incubator, water bath, or metal bath.

• Shaking improves efficiency.

• Every 5 minutes, pipette mixture again until tissue clumps are fully dissociated into single-cell suspension.

(6) Single-Cell Filtration

• Once dissociation is complete, briefly centrifuge tube for 3 seconds.

• Place tube on ice.

• Aspirate supernatant and filter through a 40 μm sterile strainer.

• Perform filtration entirely on ice.

(7) Termination of Digestion

• Add equal volume of Tissue Single-Cell Dissociation Stop Solution or complete culture medium.

(8) Cell Collection

• Centrifuge at 400 g, 4 °C for 5 minutes.

• Discard supernatant.

• Wash pellet with pre-cooled PBS, centrifuge again, discard supernatant.

(9) Red Blood Cell Lysis

• Resuspend pellet in appropriate volume of Red Blood Cell Lysis Buffer.

• Incubate on ice for 10 minutes, pipetting gently at intervals.

• Centrifuge at 300 g, 4 °C for 5 minutes, discard supernatant.

(10) Washing

• Resuspend pellet in pre-cooled PBS.

• Centrifuge at 300 g, 4 °C for 5 minutes, discard supernatant.

• Repeat wash twice.

(11) Debris Removal

• If debris remains, use High-Efficiency Debris Removal Reagent.

(12) Cell Viability Assessment

• Assess with Trypan Blue or AOPI staining.

• Viability should be >85%.

(13) Cell Cryopreservation

• If immediate downstream experiments are not possible, resuspend single cells in High-Activity Cell Cryopreservation Solution.

• Freeze directly at –80 °C for long-term storage (several years).

Notes

• For research use only.
• Perform sterile operations in a biosafety cabinet when downstream sterile culture is required.
• Enzymatic digestion must be at 37°C; all other steps should be performed on ice.
• Adjust digestion time based on tissue type and enzyme volume; minimize digestion time to avoid cell damage.
• For single-cell sequencing, accelerate the workflow and proceed immediately to library preparation after dissociation.
• Store dissolved enzyme at –20°C or –80°C for up to 6 months. Avoid more than three freeze–thaw cycles; increase enzyme volume if activity decreases.

Related:

• High-Activity Cell Cryopreservation Solution
• Red Blood Cell Lysis Buffer
• High-Efficiency Debris Removal Reagent
• Tissue Single-Cell Dissociation Stop Solution
• High-Activity Tissue Preservation Solution
• Universal Tumor Tissue Single-Cell Dissociation Enzyme
• Pancreas Tissue Single-Cell Dissociation Enzyme
• Skeletal Muscle Tissue Single-Cell Dissociation Enzyme
• Lymph Node Tissue Single-Cell Dissociation Enzyme
• Testis Tissue Single-Cell Dissociation Enzyme
• Thymus Tissue Single-Cell Dissociation Enzyme
• Neonatal Heart Tissue Single-Cell Dissociation Enzyme
• Small Intestine Tissue Single-Cell Dissociation Enzyme
• Colon Tissue Single-Cell Dissociation Enzyme
• Umbilical Tissue Single-Cell Dissociation Enzyme
• Adipose Tissue Single-Cell Dissociation Enzyme
• Retinal Tissue Single-Cell Dissociation Enzyme
• Ligament Tissue Single-Cell Dissociation Enzyme
• Embryoid Tissue Single-Cell Dissociation Enzyme
• Universal Tissue Single-Cell Dissociation Enzyme

Only for research and not intended for treatment of humans or animals

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