D001-2Enzymatic Digestion Solution Ⅰ+

Enzymatic Digestion Solution Ⅰ+

Product Description

This product is suitable for dissociating various tissues. It has high purity and good specificity, and can avoid the potential damage to cells that may be caused by other enzymes present in some trypsin extracts.

Storage

Store at -20℃ for 6 months. Store at 2-8℃ for 1 week.

Instructions for Use

(1) Take the Enzyme Digestion Solution Ⅰ+ out of the -20 °C refrigerator before use and place it at room temperature for thawing.

(2) Tissue Enzymatic Digestion

1. Cut the tissue with ophthalmic scissors. The diameter of the tissue fragments should be approximately 0.5 - 1 mm.

2. Transfer the tissue fragments into a 15 mL centrifuge tube. Centrifuge at 200 g at 2-8 °C for 5 minutes to collect the tissue.

3. After centrifugation, gently aspirate the supernatant with a Pasteur pipette without disturbing the lower sediment. Depending on the volume of the remaining tissue, add the corresponding volume of the Tissue Enzymatic Digestion Solution Ⅰ+ using a 1000 μL pipette tip (if the tissue sediment is less than 1 mL, add 5 mL of the Enzyme Digestion Solution Ⅰ+; if the tissue sediment is 1 - 2 mL, add 10 mL of the Enzyme Digestion Solution Ⅰ+). Resuspend it with a Pasteur pipette rinsed with the rinsing solution and transfer it to a new 6 cm or 10 cm culture dish (Note: During the transfer process, excessive and rapid suction is prohibited to prevent the tissue from entering the upper end of the pipette or adhering to the tube wall). Place the culture dish in a 37 °C, 5% CO₂ incubator and let it stand for enzymatic digestion.

4. During the enzymatic digestion process, take out the culture dish every 10 minutes, pipette 5 - 10 times with a Pasteur pipette rinsed with the rinsing solution for mixing (Note: During the pipetting process, excessive and rapid suction is prohibited to prevent the tissue from being sucked into the upper end of the pipette or adhering to the tube wall). Observe the degree of enzymatic digestion under a microscope (Figure 1).

Figure 1. Enzymatic digestion states of different types of tissues. Scale bar: 100 μm.

i. Under the microscope, glandular structures and cell clusters can be observed, and if there are a large number of tissue masses, enzymatic digestion needs to be continued.

ii. If there are still quite a number of incompletely digested tissue masses after 30 minutes of digestion, part of the glands and cell clusters can be collected by the natural sedimentation method. Add new Enzyme Digestion Solution Ⅰ+ to the remaining tissue masses and repeat the enzymatic digestion once (repeat steps (3) - (4)).

iii. When the clear dissociation solution becomes turbid and there are no obvious tissue masses, and more glands and cell clusters are observed under the microscope, the enzymatic digestion should be terminated.

iv. The recommended enzymatic digestion time for different tissues can refer to Table 1.

Table 1. Recommended Enzymatic Digestion Time for Different Tissues

Precautions

This product is strictly reserved for scientific research and shall not be applied to clinical diagnosis, treatment, food, or pharmaceuticals. Additionally, it is prohibited from being stored in residential premises.