Density Gradient Centrifugation: Principle and Applications

Scientists use Density Gradient Centrifugation to separate cells based on their density. Samples are placed in a centrifuge, which is a machine that spins liquid solutions at high speeds. The rotating creates a centrifugal force (gravity) in the mixture, pushing larger particles and heavier particles faster to the bottom than smaller and/or lighter particles.

Further separation according to different specific weights (density) of particles can be achieved by centrifugation of the sample mixture through a liquid of higher density as the target particle. The target particles can be collected from the top of the gradient solution, the non-wanted heavier particle will sediment through the gradient to the bottom of the tube.

Differential Centrifugation

Another centrifugation separation method based on particle mass is differential centrifugation. Because different cell sizes behave differently, the process requires no reagents or medium.

Differential centrifugation is sometimes thought to be a more basic form of centrifugation. It separates cells and organelles, whereas density gradient centrifugation separates molecules and particles. The primary distinction between the two centrifugation methods is the physical properties upon which the process is based.

Density Gradient Media

For the specific isolation of various blood cell populations using density gradient centrifugation in a single step, PluriSelect provides ready-to-use density gradient media.

PLT Spin Medium

Using a single step of centrifugation, PLT Spin Medium is a ready-to-use, sterile density medium for the high yield isolation of platelets from fresh human peripheral blood.

Monocytes Spin Medium

Monocytes Spin Medium is a ready-to-use, sterile density medium for the single step density centrifugation of monocyte enrichment in a high yield from fresh peripheral blood or buffy coat. For downstream uses like cell culture and differentiation, Monocytes Spin is a gentle substitute for bead-mediated monocyte cell enrichment.

PBMC 24+ Spin Medium

Through a single step of density gradient centrifugation, PBMC Spin 24+ Spin is a ready-to-use, sterile density medium for the isolation of PBMC from 8–24 hour old sample material (peripheral blood, boffy coat).

Density Diluent Medium

For the preparation of various density working solutions for the enrichment of cells, bacteria, and other particles, high density spin medium is a ready-to-use, sterile medium. By combining our density diluent medium with our high density spin medium, working solutions can be created.

Read More: A Brief Guide On B Cell Separation Methods And Protocols

High Density Spin Medium

For the preparation of various density working solutions for the enrichment of cells, bacteria, and other particles, density diluent medium is a ready-to-use, sterile medium. High Density Spin Medium can be diluted with Density Diluent Medium to create the working solutions.

PBMC Spin Medium

A sterile, ready-to-use density medium called PBMC Spin is used to efficiently isolate peripheral blood mononuclear cells from fresh human blood or buffy coat. A single-step density gradient centrifugation using PBMC Spin Medium can enrich cells.

Leuko Spin Medium

Leuko Spin is a sterile, ready-to-use density medium for the efficient isolation of all leukocytes from buffy coat or fresh human peripheral blood. A single-step density gradient centrifugation using Leuko Spin Medium can enrich mononuclear and polymorphonuclear (multi-lobed nuclei) cells.

Suitable Density Gradient Medium Selection

The primary function of density gradient centrifugation is to separate particles based on their buoyancy density or sedimentation rate. The following are the characteristics of an ideal density gradient media:

Sufficient solubility to produce the desired density range
Does not form high viscosity solutions in the desired density range
When the particles to be separated are osmotically sensitive, are neither hyperosmotic nor hypoosmotic.
The gradient solutions should be pH and ionic strength adjustable to be compatible with the particles being separated.
The biological activity of the sample is unaffected.
It is non-toxic and is not metabolized by cells.
It does not interfere with assay procedures and does not react with centrifuge tubes.
Displays a property that can be used to calculate concentration.
Simple to remove from the purified product
Autoclavable
Reasonable price

Unique Separation Device for Gravity Supported Cell Separation

Are you facing problems caused by poor quality sample preparation and also using outdated technology?

Our two different cell separation devices, TwinSpin, and pluriMate help to separate the target fraction regardless of whether the impurities are dense or not. The centrifugation tubes make handling easier and speed up the purification process.

PluriMate

For the best separation of leukocytes and peripheral blood mononuclear cells (PBMC) from whole blood and bone marrow, pluriMate was created. The porous sponge incorporated into the centrifuge tube’s bottom is pluriMate’s distinguishing feature. This barrier is made of premium polyurethane. You are spared the tedious and time-consuming task of overlaying the sample material. You can just pour anticoagulated blood or bone marrow straight from the blood sampling tube into the pluriMate tube. The porous barrier stops the sample material and the separation medium from mixing.Leukocytes, lymphocytes, and PBMCs are separated from unwanted erythrocytes and granulocytes during centrifugation based on their density and enriched in an interphase above the separation medium, depending on the density gradient used (Leuko Spin Medium, Lympho Spin Medium, Lympho 24+ Spin Medium, or PLT Spin Medium). After separation is finished, the barrier keeps the enriched cell fraction clean during harvest. Regardless of whether processing will be done on large or small sample volumes. There are three different tube sizes for pluriMate.

TwinSpin

TwinSpin centrifuge tubes pre-filled with PBMC Spin Medium can be used to separate peripheral blood mononuclear cells (PBMC) from whole blood and bone marrow. A standard 15 and an inner tube make up the TwinSpin. The open bottom of the inner tube is submerged in the Density Gradient Medium (DGM). Anticoagulated blood or bone marrow can be pipetted directly into the TwinSpin from the blood sampling tube.

Read More: What Is PBMC? Human PBMC Cells, PBMC Composition, and Isolation Tools

Inside the inner tube, the sample is placed on top of the DGM. Depending on the density gradient used (Leuko Spin, PBMC Spin, PBMC 24+, or PLT Spin Medium), leukocytes, lymphocytes, and PBMCs are separated from unwanted erythrocytes and granulocytes during Density Gradient Centrifugation.

As a result, the interphase is enriched in target cells above the DGM. The erythrocytes will settle out of the inner tube at the bottom of the outer tube via the DGM. Simply remove the inner tube once the separation is complete. The elastic cap serves as a valve. By removing the cap, the collection tube transforms into a pipette. Drop by drop, the contents can be collected.

Applications of TwinSpin

combines the accuracy of pipetting with density gradient cell separation to enrich cells with a high yield and maximum viability.
Useful when combined with established procedures for density gradient centrifugation
Enrichment of untouched specific cells combined with pluriSpin negative cell separation from whole blood, buffy coat, or cord blood
can be used to prepare samples for magnetic cell separation There is no need for special training or equipment.

Choose your cell separation method based on the distinguishing characteristics of your target particle and the unwanted impurities.

Reference:

Science Direct

NCBI