#densitygradient

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A buffy coat is an accumulation of platelets and white blood cells (WBCs). We'll discuss how TwinSpin helps in the preparation of a buffy coat.

A sample of peripheral whole blood contains less than 1% white blood cells (WBCs) and platelets. When scientists spin the blood sample in a centrifuge, these WBCs and platelets combine to form their own layer that is suspended between the red blood cells (RBCs) and supernatant plasma. This thin layer is referred to as a buffy coat because of its color (yellowish to brownish).

We will also find out how TwinSpin density gradient centrifugation tubes are used in buffy coat extraction.

A Brief On TwinSpin Tubes

Spin Medium can be used to separate peripheral blood mononuclear cells (PBMC) from bone marrow and whole blood into TwinSpin centrifuge tubes. A standard 15-inch tire and an inner tube make up the TwinSpin. The open bottom of the inner tube is submerged in the density gradient medium (DGM).

Instructions for Using the TwinSpin Tube

1. Ensure that the (diluted blood) sample, the TwinSpin, and the centrifuge are all at room temperature.

1.1 Check that the inner tube is partially filled and immersed in the DGM. If not, maintain the vertical position while rotating the TwinSpin device.

1.2 Take out and throw away the transport stopper.

2. Include Sample Material

2.1 Pipette the sample material on top of the DGM in the inner tube by tilting the TwinSpin 45° and allowing the sample to run down the inside of the dropper.

2.2 Push the elastic cap firmly into the opening to close the TwinSpin. PluriSpin PLT Depletion can be used to reduce platelet contamination.

3. Spin

3.1 Centrifuge at 800 x g for 20 minutes at room temperature in a swing bucket rotor, brake off. If the blood is more than 4 hours old, centrifuge it for 30 minutes at 1000g.

4. Collect

4.1 Remove the inner cell separation tube by unscrewing it.

4.2 Push the elastic cup down to collect cells in the opaque layer in a new tube.

5 Wash (if necessary)

5.1 Add wash buffer to the reaction tube.

5.2 Cells are spun down for 10 minutes with 300 x g at 4 °C (no or small break).

5.3 Remove the supernatant and set the reaction tube on the table for 20 seconds.

Excess wash buffer will flow down the tube wall and accumulate at the bottom. Aspirate the majority of the liquid above the pellet. The liquid will appear foggy because it contains mostly platelets; aspiration will improve the washing result.

5.5 Carefully pipette the pellet with 1 ml of wash buffer.

5.6 Re-do steps 5.1–5.4.

5.7 Refill the pellet to the desired volume.

Preparation of buffy coat from scratch

TwinSpin centrifuge tubes support density gradient centrifugation. Let’s find out how and we will also discuss the protocol for the use of TwinSpin in combination with density gradient media.

Scientists divide materials using the centrifugation technique based on their size and shape. The two types of centrifugal methods used to separate particles are differential centrifugation and density gradient centrifugation. This blog will discuss TwinSpin's functionality as well as how it supports density gradient centrifugation.

Peripheral blood mononuclear cells (PBMC) can be separated from whole blood and bone marrow using TwinSpin centrifuge tubes that have already been pre-filled with PBMC Spin Medium. The TwinSpin is made up of an inner tube and a standard 15. The inner tube's open bottom is buried in the density gradient medium (DGM). The blood sampling tube can be directly pipetted into the TwinSpin with anticoagulated blood or bone marrow. 

The sample is placed on top of the DGM inside the inner tube. 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. 

Target cells above the DGM are therefore enriched in the interphase. Through the DGM, the erythrocytes will exit the inner tube and fall to the bottom of the outer tube. Once the inner tube has completely separated, just take it out. 

As a valve, the elastic cap is used. The collection tube can be converted into a pipette by removing the cap. Drop by drop, the contents can be collected.

Instructions for Using the TwinSpin Tube

1. Ensure that the (diluted blood) sample, the TwinSpin, and the centrifuge are all at room temperature.

1.1 Check that the inner tube is partially filled and immersed in the DGM. If not, maintain the vertical position while rotating the TwinSpin device.

1.2 Take out and throw away the transport stopper

2. Include Sample Material

2.1 Pipette the sample material on top of the DGM in the inner tube by tilting the TwinSpin 45° and allowing the sample to run down the inside of the dropper.

2.2 Push the elastic cap firmly into the opening to close the TwinSpin. PluriSpin PLT Depletion can be used to reduce platelet contamination.

3. Spin

3.1 Centrifuge at 800 x g for 20 minutes at room temperature in a swing bucket rotor, brake off. If the blood is more than 4 hours old, centrifuge it for 30 minutes at 1000g.

4. Collect

4.1 Remove the inner separation tube by unscrewing it.

4.2 Push the elastic cup down to collect cells in the opaque layer in a new tube.

5 Wash (if necessary)

5.1 Add wash buffer to the reaction tube.

5.2 Spin down cells for 10 minutes at 4°C with 300 x g (no or small brake).

5.3 Remove the supernatant and set the reaction tube on the table for 20 seconds.

Excess wash buffer will flow down the tube wall and accumulate at the bottom. Aspirate the majority of the liquid above the pellet. The liquid will appear foggy because it contains mostly platelets; aspiration will improve the washing result.

5.5 Carefully pipette the pellet with 1 ml of wash buffer.

5.6 Re-do steps 5.1–5.4.

5.7 Refill the pellet to the desired volume.