Dispase targets fibronectin, collagen IV, and to a lesser extent collagen I. These are extracellular matrix proteins that are important for cell adhesion and tissue structure. By cleaving these proteins, dispase disrupts cell-cell and cell-matrix interactions, allowing tissues to be dissociated into single cells. The enzyme's specificity for fibronectin and collagen IV makes it useful for the dissociation of tissues without damaging cell surface proteins. This is particularly important for preserving cell surface markers for flow cytometry and other applications. Dispase is a neutral protease, meaning it is active at physiological pH, which makes it suitable for use with living cells. The enzyme's activity can be inhibited by EDTA, EGTA, and other metal chelators. It is also inhibited by serum, so it is typically used in serum-free media.

Instruction for use
1. Solution preparation
1) Dissolve an appropriate amount of freeze-dried powder of this product in DPBS buffer salt solution (without calcium and magnesium ions), to prepare a stock solution of 10 mg/mL, and filter it through a 0.22 μ M filter membrane for sterilization.
2) Dilute the above stock solution with DPBS to the working solution for use. The commonly used working solution concentration for cell separation is 0.6-2.4 U/mL.
Note: It is not recommended to use working concentrations higher than 2.4 U/mL.
2. Dissection/dissociation of tissues
1) Cut the tissues into 3-4 mm tissue blocks using sterile knives or scissors;
2) Wash tissues with sterile PBS;
3) Add Disperse II solution (working concentration of 0.6-2.4 U/mL) to the tissue block and ensure that the tissues are completely immersed in the Disperse solution.
4) Incubate at 37 ℃ and slowly stir until all tissues are dissociated.
Note: Generally, for tissues that are difficult to dissociate, the separation goal can be achieved within 1 hour, but longer incubation times (such as several hours) will not significantly affect cell activity.
5) If necessary, the above digested products can be filtered through a sterile stainless steel mesh to separate single cells from residual tissue blocks. Alternatively, after the large tissue has settled, gently pour out the upper layer of cells. If necessary, use fresh dispase solution to further dissociate the residual tissue.
6) Centrifuge and precipitate the cells, then discard the enzyme solution;
7) Resuspend the cell pellet in culture medium and culture the cells under normal conditions.
3. Cell passage
1) Immerse the cells in Dispase solution (preheated at 37 ℃) and incubate at 37 ℃ for 5 minutes;
2) Remove the above solution and continue incubating at 37 ℃ for 10 minutes;
3) Observe cell separation under a microscope, and if necessary, further incubate for 15 minutes;
4) Suspend cells in cell culture medium and gently rotate to precipitate the cells. Wash the cells with the culture medium;
5) Resuspend the cells in fresh cell culture medium and plate the cells under normal conditions.

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In vitro, dispase is used for the dissociation of tissues and cells in cell culture. It is used for the preparation of primary cell cultures from embryonic and neonatal tissues, as well as from adult tissues. The enzyme is added to tissue samples in serum-free media and incubated at 37°C for 15-60 minutes. The tissue is then mechanically dissociated by pipetting or by using a cell strainer. The resulting cell suspension contains single cells that can be used for cell culture, flow cytometry, or other applications. Dispase is also used for the detachment of cells from culture surfaces. It is particularly useful for detaching cells that are difficult to detach with trypsin, such as epithelial cells and stem cells. The enzyme's gentleness makes it suitable for use with sensitive cell types. Dispase is also used in studies of cell adhesion and migration.

The technical difficulty to isolate microglia, astrocytes and infiltrating immune cells from mouse brain is nowadays a limiting factor in the study of neuroinflammation. Brain isolation requirements are cell-type and animal-age dependent, but current brain dissociation procedures are poorly standardized. This lack of comprehensive studies hampers the selection of optimized methodologies. Thus, we present here a comparative analysis of dissociation methods and Percoll-based separation to identify the most efficient procedure for the combined isolation of healthy microglia, astrocytes and infiltrated leukocytes; distinguishing neonatal and adult mouse brain. Gentle mechanical dissociation and DNase I incubation was supplemented with papain or collagenase II. Dispase II digestion was also used alone or in combination. In addition, cell separation efficiency of 30 % and 30-70 % Percoll gradients was compared. In these experiments, cell yield and integrity of freshly dissociated cells was measured by flow cytometry. We found that papain digestion in combination with dispase II followed by 30 % Percoll separation is the most balanced method to obtain a mixture of microglia, astrocytes and infiltrated immune cells; while addition of dispase II was not an advantage for neonatal brain. These dissociation conditions allowed flow cytometry detection of a slight glial activation triggered by sublethal LPS injection. In conclusion, the enzymes and Percoll density gradients tested here affected differently resting microglia, activated microglia/macrophages, astrocytes and infiltrated lymphocytes. Also, newborn and adult brain showed contrasting reactions to digestion. Our study highlights the strength of flow cytometry for the simultaneous analysis of neuroimmune cell populations once extraction is optimized[2].

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In vivo, dispase is not typically used as a therapeutic agent. It is primarily used as a research tool for tissue dissociation and cell culture. However, the enzyme has been studied for its potential use in wound healing and tissue repair, as it can help to remove damaged tissue and promote the growth of new tissue. Dispase has also been used in studies of skin grafting and burn wound healing. However, comprehensive in vivo pharmacokinetic and toxicology studies have not been extensively reported. The enzyme is classified as a research chemical and is not approved for human use as a therapeutic agent. Further in vivo studies are needed to fully characterize the enzyme's therapeutic potential and safety profile.

In vitro enzyme assays for dispase typically involve the use of a chromogenic or fluorogenic substrate. A common substrate is casein, which is hydrolyzed by dispase to release peptides. The activity of the enzyme is measured by the increase in absorbance or fluorescence. Another common substrate is azocoll, a collagen-based substrate that releases azo dye upon hydrolysis. The enzyme is incubated with the substrate in an appropriate buffer (e.g., Tris-HCl, pH 7.4-8.0) at 37°C for a specified time. The reaction is terminated by the addition of trichloroacetic acid or by cooling on ice. The released peptides or dye are then measured spectrophotometrically. One unit of dispase activity is defined as the amount of enzyme that hydrolyzes a specified amount of substrate per minute under the assay conditions. Inhibition studies are conducted by pre-incubating the enzyme with potential inhibitors before adding the substrate.

Dispase, a neutral protease isolated from culture filtrates of Bacillus polymyxa, has proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis and intact epithelial sheets in culture from the substratum. In both cases it effects separation by cleaving the basement membrane zone region while preserving the viability of the epithelial cells. Because it is not known what or where in the basement membrane zone Dispase cleaves, we set up studies to define its substrate specificity. Using purified basement membrane components and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we show that Dispase cleaves fibronectin and type IV collagen, but not laminin, type V collagen, serum albumin, or transferrin. The action of Dispase on collagen appears to be selective for type IV collagen in that several stable degradation products are formed, whereas the enzyme degrades type I collagen only minimally. In newborn human skin, as seen by electron microscopy, Dispase removes the lamina densa, rich in type IV collagen, but preserves the anchoring fibrils (structures known to contain type VII collagen) and the epidermal cells. Because its action is so selective, it suggests that Dispase can serve as a powerful tool for dissecting epithelial-mesenchymal interactions[1].

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In vitro cell-based assays for dispase are performed using cell culture models to study its effects on cell dissociation and adhesion. Cells are cultured in appropriate medium and treated with dispase at various concentrations (typically 0.1-2.4 U/mL) for varying periods. Following treatment, cell detachment is assessed by microscopy or by counting the number of cells in suspension. Cell viability is assessed using trypan blue exclusion or by measuring metabolic activity (e.g., MTT). For studies of cell adhesion, cells are allowed to attach to culture surfaces in the presence or absence of dispase, and the number of attached cells is quantified. Each experiment includes appropriate controls (untreated cells, vehicle controls) and is performed in triplicate to ensure statistical reliability.

In vivo animal experiments with dispase are limited. The enzyme has been studied in animal models of wound healing and tissue repair. In these studies, dispase is applied topically to wounds to remove damaged tissue and promote the growth of new tissue. The enzyme's effects on wound healing are assessed by measuring wound closure rates, tissue histology, and markers of tissue repair. All animal procedures are conducted in accordance with institutional animal care and use committee guidelines, with appropriate sample sizes (typically n=6-10 per group) to ensure statistical power.

The pharmacokinetic properties of dispase are not applicable, as the enzyme is primarily used as a research tool and is not administered systemically. If administered systemically, the enzyme would likely be rapidly cleared from circulation by the immune system and would be degraded by proteases. The enzyme is active over a broad pH range (pH 5.5-8.5) and is stable at temperatures up to 40°C. It is inhibited by EDTA, EGTA, and other metal chelators, and by serum. The enzyme's activity can be influenced by the presence of these inhibitors.

The toxicological profile of dispase is generally favorable, as the enzyme is used for cell culture and tissue dissociation without significant toxicity to cells at the concentrations typically used. The enzyme is a bacterial neutral protease and is not expected to be toxic at low concentrations. However, high concentrations of dispase can damage cells by over-digesting cell surface proteins. The enzyme is classified as a research chemical and is not approved for human use. Standard safety precautions should be observed when handling the enzyme, including the use of appropriate personal protective equipment. As with all enzymes, inhalation and skin contact should be avoided.

[1]. Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. J Invest Dermatol. 1989 Aug;93(2):287-90.

[2]. Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry. IBRO Rep. 2020 Jan 13;8:36-47.

Dispase is a valuable research tool for cell culture, tissue engineering, and stem cell research. It is a bacterial neutral protease enzyme that is used for the dissociation of tissues and cells in cell culture. Dispase cleaves fibronectin, collagen IV, and to a lesser extent collagen I. It is used as a gentle but effective enzyme for the dissociation of tissues into single cells for primary cell culture. The enzyme is particularly useful for the dissociation of embryonic and neonatal tissues, as well as for the detachment of cells from culture surfaces. Dispase is also used for the preparation of cell suspensions for flow cytometry and other applications. The enzyme is supplied as a lyophilized powder or as a sterile solution. Dispase is not approved for any clinical indication and is strictly for research use only. Its role as a tissue dissociation enzyme makes it an essential tool for cell culture and biomedical research.

42613-33-2

White to off-white solid powder

Neutral protease, Paenibacillus polymyxa

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 25 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)