1. Sedimentation Coefficient
The resolution in centrifugation is influenced by the ratio of the zone width to the radial path. A higher sedimentation coefficient results in a greater distance between cell zones, allowing for better separation. When cells have similar sedimentation coefficients, it becomes more challenging to distinguish them. To improve resolution, the initial cell suspension should be as narrow as possible, and the sample volume should be minimized. Additionally, using a longer gradient column—such as a long centrifuge tube—helps compensate for differences in sedimentation coefficients and reduces adverse effects during the process.
2. Gradient Density Range
The density of the cells must exceed the lowest density of the gradient medium. It is recommended to use gradients with both high and low viscosity to ensure efficient and rapid separation. This helps achieve better resolution and minimizes the time required for the process.
3. Gradient Volume
The volume of the gradient varies depending on the separation device used. Typically, it ranges from 5 ml to 1600 ml. The size of the rotor and the recovery method also influence the gradient volume. Unlike isopycnic centrifugation, speed sedimentation requires careful consideration, as very few gradient media are suitable for this technique.
4. Cell Loading
Cell loading is crucial because cell interactions or aggregation depend on concentration. Two key issues arise: determining how many cells to add to achieve the desired resolution and ensuring the concentration is low enough to prevent aggregation while maintaining sufficient cell recovery and purity. Due to variability among cell types from different tissue sources, no universal method exists. Most approaches rely on experience or published protocols, which may have limitations in practical applications.
5. Centrifugal Speed and Time
Choosing the optimal centrifugal speed depends on factors such as cell size, density, and the viscosity and density range of the medium. The applied centrifugal force must be strong enough to allow cells to move through the gradient and reach their equilibrium positions. If the speed is too low, the time required for separation increases, potentially leading to cell damage, especially in high-viscosity media. For example, prolonged centrifugation of cardiomyocytes can cause oxygen deprivation and cell death. Conversely, if the speed is too high, smaller cells may pass through the gradient too quickly, mixing with larger cells. Therefore, balancing speed and time is essential. To minimize instability or loss of activity, faster separations using higher speeds are often preferred. In cases where larger cells settle too slowly, a highly viscous gradient medium can help control their settling rate.
6. Stability
Most cell isolation procedures are performed at room temperature (20–25°C), although some may be carried out at 4°C when necessary. Maintaining stable conditions ensures the viability and functionality of the isolated cells.
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