Cell Separation Medium (Density Gradient)
Based on 1 Customer Validation
MCE Cell Separation Medium (Density Gradient) is a low-density gradient medium characterized by its broad applicability, operational simplicity, and gentle separation performance.It is primarily composed of silica particles (15-30 nm in diameter) coated with a monolayer of polyvinylpyrrolidone (PVP).
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Storage :
4°C, 5 years. Unopened: Store at 4-30°C. After opening: It is recommended to store the reagent at 4°C under sterile conditions. If stored under non-sterile conditions, the reagent should be frozen at -20°C to prevent microbial growth (ensure sufficient expansion space to avoid container rupture). The maximum storage period is 6 months. Note: After freezing and thawing, the separation medium may exhibit density gradients or phase separation. Mix thoroughly before use to ensure homogeneity and experimental reliability.
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Description & Advantages
MCE Cell Separation Medium (Density Gradient) is a low-density gradient medium characterized by its broad applicability, operational simplicity, and gentle separation performance. It is primarily composed of silica particles (15-30 nm in diameter) coated with a monolayer of polyvinylpyrrolidone (PVP). Due to the heterogeneity in particle size, the particles sediment at different rates during centrifugation, spontaneously forming a continuous, homogeneous, and isotonic gradient with a density range of 1.0-1.3 g/mL.
This medium is suitable for the separation of cells, subcellular organelles, bacteria, and viruses, and can also effectively distinguish intact viable cells from damaged or fragmented cells.
Features of MCE Cell Separation Medium (Density Gradient)
1. Wide separation range: Enables efficient separation of biological particles with sedimentation coefficients greater than 60 S.
2. Mild and non-destructive: Allows gentle isolation of subcellular components and large viral particles (~70 S) while maintaining biological activity and structural integrity.
3. Physiological compatibility: Adjustable to physiological ionic strength and pH for biologically relevant separations.
4. Non-toxic and chemically inert: Exhibits no cytotoxicity and does not adhere to or react with cell membranes.
5. Isotonic gradient formation: Generates a self-forming isotonic density gradient ranging from 1.0 to 1.3 g/mL, ensuring stable morphology and viability of cells.
6. Flexible centrifugation options: Suitable for both pre-formed and self-generated gradients under moderate-speed centrifugation in an angle rotor.
7. Low viscosity: Viscosity of 10 ± 5 cP at 20°C enables rapid gradient formation and efficient particle separation.
8. Excellent stability: Stable for up to 5 years at room temperature when unopened, and for up to 2 years after opening under sterile conditions.
9. Autoclavable: Undiluted solution can be re-sterilized by autoclaving at 120°C for 30 minutes without performance loss.
Protokoll
Preparation of Separation Solutions with Different Densities
To obtain the desired density gradient, the stock separation medium can be diluted with an appropriate buffer such as physiological saline or sucrose solution.
| Application | Dilution system/condition |
|---|---|
| Gradient separation of cell samples | PBS may be used as the diluent |
| Gradient separation of subcellular particles | 0.25 M sucrose is recommended as the diluent |
| Isotonic separation solution | 9 parts stock + 1 part 1.5 M NaCl or 2.5 M sucrose |
| Lower-density dilution for cell separation | 0.15 M NaCl or 1× cell culture medium |
| Lower-density dilution for subcellular/viral particles | 0.25 M sucrose |
Preparation of Isotonic Separation Solution
1. Mix 9 parts of stock separation medium (v/v) with 1 part of 1.5 M NaCl or 2.5 M sucrose solution (v/v) thoroughly to obtain an isotonic separation solution.
2. Further adjust the osmotic pressure to the desired value by adding salt ions or distilled water. An osmometer is recommended for calibration and reproducibility.
3. The stock density ρ0 is 1.13 ± 0.005 g/mL (refer to COA for the exact value). The density of 1.5 M NaCl is 1.058 g/mL and that of 2.5 M sucrose is 1.316 g/mL. If ρ0 = 1.13 g/mL, the isotonic separation solution density is approximately 1.123 g/mL in 1.5 M NaCl and 1.149 g/mL in 2.5 M sucrose.
Dilution of Isotonic Separation Solution to Lower Densities
1. For cell separation, dilute the isotonic separation solution to the desired density using 0.15 M NaCl or 1× cell culture medium.
2. For subcellular or viral particle separation, dilute using 0.25 M sucrose.
3. Example: If 55 mL of isotonic separation solution (ρi = 1.123 g/mL) is used and the desired final density is 1.07 g/mL, 44.6 mL of 0.15 M NaCl is required.
Note: The calculated density generally approximates the target value. For precise control, measure the actual density with a densitometer or refractometer.
One-Step Dilution Method for Preparing Separation Solution at the Desired Density
1. In a graduated cylinder, first add 1.5 M NaCl or 2.5 M sucrose solution to one-tenth of the final desired volume.
2. Calculate the required stock volume using the Manual formula: V0 = V × (ρ - 0.1ρ10 - 0.9) / (ρ0 - 1).
3. Add the calculated stock volume and then add distilled water to reach the target final volume.
4. Example: To prepare 100 mL of separation solution at 1.07 g/mL in 0.15 M NaCl, assuming ρ0 = 1.13 g/mL, first add 10 mL of 1.5 M NaCl, then 49.4 mL of stock separation medium, and finally add distilled water to 100 mL.
Preparation and Application of Discontinuous Density Gradient Separation Solutions
1. Prepare a series of separation solutions of different densities using the methods above.
2. Centrifuge Tube Pretreatment: Rinse the inner wall of the centrifuge tube with bovine serum and gently remove any excess liquid.
3. Assembly of the Discontinuous Density Gradient: Carefully layer the separation solutions from highest to lowest density along the tube wall, avoiding splashing or mixing between layers.
4. Sample Loading: Gently add the sample onto the assembled density gradient. Adjust sample volume and cell concentration according to sample type and avoid excessive loading.
5. Centrifugation: Perform centrifugation at 400 × g for 20-25 min using a slow and smooth acceleration/deceleration program.
6. Fraction Collection: If target cells are at the interface between two layers, carefully remove the upper layer and collect the interface. If they are distributed within a specific layer, collect the corresponding fraction sequentially by density.
7. Optional Washing: For cell samples, wash the viable fraction 2-3 times with physiological saline or PBS at a 5 : 1 volume ratio and centrifuge at 200 × g for 2-10 min. For viral or subcellular samples, centrifuge at 100,000 × g for 2 h with a swinging-bucket rotor or at 100,000 × g for 90 min with a fixed-angle rotor; the separation matrix pellets while the target sample remains in the supernatant.
Speicherung
4°C, 5 years.
Unopened: Store at 4-30°C.
After opening: It is recommended to store the reagent at 4°C under sterile conditions. If stored under non-sterile conditions, the reagent should be frozen at -20°C to prevent microbial growth (ensure sufficient expansion space to avoid container rupture). The maximum storage period is 6 months.
Note: After freezing and thawing, the separation medium may exhibit density gradients or phase separation. Mix thoroughly before use to ensure homogeneity and experimental reliability.
Achtung
1. It is recommended to use polycarbonate (PC) centrifuge tubes when performing centrifugation with this product. During centrifugation, silica particles may aggregate at the bottom of the tube or at the phase interface, and may adhere to the tube wall. Once dried, these residues can be difficult to remove; therefore, it is advised to rinse all related equipment immediately after use with deionized water to prevent residue buildup.
2. The product remains chemically stable within the pH range of 5.5-10 and is not affected by mild variations in pH. However, gelation may occur below pH 5.5. In addition, the presence of divalent cations or elevated temperatures can further promote gelation.
3. The product cannot be sterilized by filtration. If autoclaving is required, it should be performed only on stock solutions free of salts and sucrose. The presence of salts may induce gelation, while sucrose may caramelize under heat. During autoclaving, minimize air exposure by using narrow-neck containers to reduce solid particle formation. If particles form after sterilization, they can be removed by filtration or low-speed centrifugation. Should volume loss occur after sterilization, the lost volume may be restored with distilled water without affecting the density of the solution.
4. This product is for R&D use only, not for drug, household, or other uses.
5. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Components | HY-K3023-100 mL | HY-K3023-500 mL |
|---|---|---|
| Cell Separation Medium (Density Gradient) | 100 mL | 100 mL × 5 |
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