Is it sterile: Yes
Universal Cell Separation Medium is a fully validated density gradient centrifugation medium, suitable for the separation of cells, viruses, and subcellular particles. Based on well-established density gradient centrifugation technology, this product enables gentle, rapid, and efficient separation of a wide variety of cells, viruses, and subcellular components within the density range of 1.0–1.3 g/mL, and is applicable to most biological particle separation experiments with sedimentation coefficients greater than 60S.
The core component of this product is polyvinylpyrrolidone (PVP)-coated modified silica gel particles, with a particle size of 15–30 nm and a free PVP content of only 1%–2%, ensuring a stable and reliable formulation. Due to the inherent size variation of silica gel particles, differential sedimentation occurs during centrifugation, spontaneously generating a continuous isotonic density gradient. The experimental procedure is flexible and convenient: density gradients can be prepared using a gradient mixer or high-speed centrifuge; alternatively, the sample can be pre-mixed with the medium, allowing the gradient to form in situ and achieving both gradient establishment and sample separation in a single step. Target components can be obtained in one operation, effectively simplifying the experimental workflow and improving efficiency.
Product Applications
This product is suitable for processing biological samples such as blood and tissues. It efficiently performs the separation, purification, and enrichment of cells, bacteria, viruses, organelles, and various subcellular components. It also effectively separates intact viable cells from damaged cells and cellular debris.
Product Features
● Excellent Activity Protection: Low viscosity, non-toxic at isotonic conditions, high biocompatibility. Does not penetrate biological membranes, does not adhere to cells, and effectively preserves the density and viability of biological particles throughout the separation process.
● Convenient and Efficient Use: Spontaneously forms a continuous density gradient upon centrifugation. Supports large-volume sample processing with simple operation and short processing time.
● Broad Applicability: Compatible with a wide range of commonly used buffers and culture media, allowing flexible preparation of density gradients within the range of 1.0–1.3 g/mL.
● Stable and Reliable Performance: Optimized formulation with stringent quality control. Exhibits stable physicochemical properties. The stock solution can be repeatedly autoclaved, ensuring excellent experimental reproducibility.
Physical Properties and Specifications
● Appearance: Colorless to light yellow liquid
● Density: 1.130 ± 0.005 g/mL
● Osmolality: max. 25 mOsm/kg·H₂O
● Conductivity: max. 100 mS/m
● pH: 9.0 ± 0.5
● Viscosity: max. 15 cP
Instructions (For Reference Only)
1. Preparation of Density Gradient Medium
This product is based on the principle of density gradient centrifugation for sample separation. Separation is achieved by exploiting the differential sedimentation rates of various cells and particles in media of different densities. Therefore, the accuracy of the working solution density is the key factor ensuring successful separation. In addition to precise density control, appropriate osmolality must be maintained to preserve the structure and viability of the sample to be separated.
Universal Cell Separation Medium performs optimally in balanced salt solutions, physiological saline, or 0.25 M sucrose solutions. For cell samples, separation can be performed in gradients prepared with balanced salt solutions, cell culture media, or NaCl solutions. For subcellular components or viruses, which tend to aggregate in salt-containing environments, gradients prepared with sucrose solutions are recommended.
Note: When preparing gradients, you may directly follow the volume calculated in Step 1.1, mix the appropriate volumes of each solution to obtain the desired density, and then proceed to Step 1.3 for gradient preparation. Alternatively, you may first prepare an isotonic stock solution according to Step 1.2, dilute it with the isotonic solution to the desired density, and then proceed to Step 1.3 for gradient preparation.
1.1 One-Step Dilution (Direct Preparation of Target Working Solution)
(1) Determine the target density and volume of the working solution based on the target separation components. Then calculate the required volumes of undiluted Universal Cell Separation Medium, diluent, and distilled water using the following formula:
V₀ = V × (ρ − 0.1ρ₁₀ − 0.9) / (ρ₀ − 1)
V₀ = Volume of undiluted Universal Cell Separation Medium (mL)
V = Total volume of target working solution (mL)
ρ = Target density of working solution (g/mL)
ρ₀ = Density of undiluted Universal Cell Separation Medium (1.130 g/mL)
ρ₁₀ = Density of the diluent (density of 1.5 M NaCl solution is approximately 1.058 g/mL)
(2) After calculating V₀ using the above formula, measure V/10 of the diluent (1.5 M NaCl solution or 2.5 M sucrose solution). Mix the Universal Cell Separation Medium and the diluent thoroughly, then bring the final volume to V with distilled water.
Example: To prepare 100 mL of target working solution with a density of 1.07 g/mL using 1.5 M NaCl solution as the diluent:
ρ = 1.07 g/mL, ρ₀ = 1.130 g/mL, ρ₁₀ = 1.058 g/mL, V = 100 mL
Substituting into the formula: V₀ = 100 × (1.07 − 0.1 × 1.058 − 0.9) / (1.130 − 1) = 49.4 mL
Procedure: Mix 49.4 mL of Universal Cell Separation Medium with 10 mL of 1.5 M NaCl solution, then add distilled water to a final volume of 100 mL to obtain the target working solution.
1.3 Preformed Density Gradient Preparation:
Starting from the bottom to the top, layer the solutions in order of decreasing density (from high to low). Using a pipette tip or syringe, position it against the tube wall and allow the liquid to flow down naturally and slowly. Ensure that the upper layer (low density) does not splash into the lower layer (high density) or mix at the interface, so as to form a clean gradient.
Note: The tube wall may be pre-rinsed with FBS, and excess serum then removed. This pretreatment allows the layered separation solutions to flow smoothly along the tube wall, resulting in well-defined interfaces.
2. Density Gradient Centrifugation
The appropriate centrifugation method can be selected according to experimental requirements.
2.1 High-Speed Angle-Rotor Centrifugation (Spontaneous Gradient Formation):
Universal Cell Separation Medium will spontaneously form a continuous density gradient from the bottom to the top of the tube after centrifugation at approximately 10,000 × g (prepared in 0.15 M physiological saline) or 25,000 × g (prepared in 0.25 M sucrose solution) for 15 minutes. In this method, the sample can be uniformly mixed with the Universal Cell Separation Medium prior to centrifugation. During centrifugation, sample particles will be stratified within the spontaneously formed gradient according to their densities, and will ultimately become concentrated at the position where their density equals that of the surrounding medium, forming distinct isopycnic bands. This method is suitable for the efficient separation of subcellular particles or virus samples.
2.2 Low-Speed Swing-Bucket Rotor Centrifugation (Dependent on Preformed Gradients):
For shear-sensitive samples such as viable cells, low-speed centrifugation (e.g., 400 × g) using a swing-bucket rotor is recommended. This method typically relies on preformed continuous or discontinuous density gradients (Step 1.3). The sample can be gently layered onto the separation medium using a pipette tip or syringe before centrifugation. The centrifugation should be performed with slow acceleration and deceleration settings (e.g., set to grade 0 or 1) for 20–30 minutes. This approach effectively maintains gradient stability and prevents the already separated cell bands from remixing due to fluid disturbance, thereby allowing cells to migrate gently to their isopycnic positions, resulting in higher cell viability and recovery rates.
3. Harvesting Target Components
After density gradient centrifugation, the system forms a continuous density gradient (approximately 1.0–1.3 g/mL), in which various cells or particles will float or sediment to isopycnic bands where their own density (i.e., buoyant density) equals that of the surrounding medium. The target components can be obtained by carefully aspirating the target layer bands.
The typical densities and distribution positions of different cells and subcellular components in Universal Cell Separation Medium are provided in the table below for reference only. Please note that variations may exist among samples from different species.
| Category | Sample Type | Buoyant Density (g/mL) | Position After Separation | Remarks | | Cells | Platelet | <1.04 | Upper layer or floating on liquid surface | Lightest, often floats on the surface | | Cells | Lymphocyte | 1.055-1.065 | Middle layer, interface | Often aggregates at the transparent band | | Cells | Monocyte | 1.060-1.068 | Middle-lower layer | Located below lymphocytes | | Cells | Neutrophil | 1.080-1.090 | Lower layer | Directly above red blood cells | | Cells | Erythrocyte | >1.09 | Bottom pellet | Highest density, completely sediments to the bottom | | Subcellular Components | Cytoplasm | <1.05 | Upper layer or floating | Lightest soluble fraction | | Subcellular Components | Microsome | 1.09-1.12 | Middle layer | Broad distribution | | Subcellular Components | Lysosome | 1.12-1.15 | Middle-lower layer | Often located above mitochondria | | Subcellular Components | Mitochondria | 1.17-1.20 | Lower layer | Mainly concentrated in higher density region | | Subcellular Components | Nucleus | >1.20 | Very bottom | Highest density among nuclei |
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4. Washing
Since Universal Cell Separation Medium is non-cytotoxic and does not adhere to cell membranes, it is generally not necessary to remove it. Cells can be directly transferred to culture systems, viruses can be directly used for infection, and organelles can be directly applied to functional studies. If removal is required, washing can be performed according to the following methods.
(1) Cell samples: Gently wash with 5 volumes of pre-chilled physiological saline or PBS. Centrifuge at 200–400 × g for 2–10 minutes at 4°C. Discard the supernatant. Repeat washing 2–3 times if necessary, and collect the cell pellet.
(2) Subcellular components: After gentle washing with 5 volumes of pre-chilled physiological saline or PBS, centrifuge at 10,000–20,000 × g for 10–30 minutes at 4°C. Carefully discard the supernatant and collect the pellet.
Note: The specific centrifugal force depends on the target subcellular components.
(3) For viruses, exosomes, or smaller particles: For undiluted samples, centrifuge at 100,000 × g for 120 minutes (swing-bucket rotor) or 90 minutes (angle rotor) at 4°C to pellet the medium components from the Universal Cell Separation Medium, and collect the supernatant.
(4) Washing can also be performed by gel filtration, ion exchange chromatography, ultrafiltration centrifugation, or dialysis according to published protocols.
5. Reference Data: Density Determination of Universal Cell Separation Medium Gradients
A refractometer can be used to measure the density of fractionated Universal Cell Separation Medium solutions. The refractive index shows a linear correlation with the density of Universal Cell Separation Medium solutions. For reference, the density and refractive index of dilution series of Universal Cell Separation Medium in sucrose and sodium chloride solutions at 20°C are provided in the table below.
| Density in Sucrose Solution (g/mL) | Refractive Index in Sucrose Solution | Density in NaCl Solution (g/mL) | Refractive Index in NaCl Solution | | 1.0345 | 1.3457 | 1.0085 | 1.335 | | 1.0484 | 1.3478 | 1.0243 | 1.3372 | | 1.0618 | 1.3499 | 1.0403 | 1.3399 | | 1.0765 | 1.3518 | 1.0558 | 1.3423 | | 1.0903 | 1.3541 | 1.0713 | 1.3449 | | 1.104 | 1.3561 | 1.0869 | 1.347 | | 1.118 | 1.3582 | 1.1029 | 1.3493 | | 1.1319 | 1.36 | 1.1189 | 1.3519 | | 1.1461 | 1.3626 | 1.1305 | 1.3534 | | 1.1547 | 1.3638 | 1.1513 | 1.3569 |
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Precautions
1.After centrifugation, this product may leave residual silica precipitates on the tube wall and bottom, which are difficult to remove once dried. Therefore, consumables should be cleaned immediately after the experiment. Polycarbonate (PC) centrifuge tubes are recommended.
2.This product is stable within the pH range of 5.5–10.0. Conditions such as pH < 5.5, the presence of divalent cations (e.g., Ca²⁺, Mg²⁺), or high temperatures may cause colloidal silica gelation and destabilization. Experimental systems should avoid low pH, high salt, and divalent ion-containing environments.
3.Aggregation of colloidal silica upon long-term storage is an inherent property. In some batches, aggregates may be observed as slight precipitates at the bottom of the container or as a faint whitish band at a density of 1.04–1.05 g/mL. This band may form during gradient preparation centrifugation or low-speed centrifugation of preformed gradients. Silica aggregates do not interfere with biological particle separation, as the buoyant density of most cells and organelles in the separation medium is greater than 1.05 g/mL. For routine cell, virus, and organelle separation experiments, the impact of silica aggregates from the gradient medium is negligible.
4.This product is sterile-packaged. Strict aseptic techniques must be followed during operation to prevent contamination. If re-sterilization is required, only the stock solution can be autoclaved at 121°C for 30 minutes. Pre-mixed working solutions containing salts or sucrose should not be autoclaved, as this may induce gelation or caramelization; filtration through 0.22 μm membrane filters is also not recommended. During sterilization, minimize contact between the liquid and air to avoid solid particle precipitation at the liquid-air interface; narrow-mouth containers are recommended. If particles form, they can be removed by filtration through a 0.45 μm filter or by low-speed centrifugation at 500 × g for 5 minutes. If liquid evaporates during sterilization, the original density can be restored by adding the corresponding volume of distilled water.
5.Laboratory coats and disposable gloves should be worn during operation. Ensure proper safety protection.
6.This product is for research use only.
