15 mL / 50 mL Lymphocyte Separation Tube(Empty), with Porous Barrier, for PBMC Isolation

Description

The cell separation tube is designed for the isolation of lymphocytes and mononuclear cells from whole blood. A porous sieve plate is integrated inside the tube to prevent premature mixing of the blood sample with the separation medium below the barrier, thereby reducing contamination of upper-layer cells by erythrocytes and facilitating easier recovery of the separated cells.

This product utilizes the density gradient centrifugation method. Based on differences in cell density, a separation medium and centrifugation are applied to achieve cell separation and purification. The density gradient separation medium forms a defined gradient after loading anticoagulated whole blood into the tube. Following centrifugation, erythrocytes and granulocytes sediment to the bottom of the tube, while PBMCs (peripheral blood mononuclear cells, including lymphocytes and monocytes) remain at the interface above the separation medium. A small fraction of cells may remain suspended within the medium. Lymphocytes can be collected by aspirating the cell layer at the interface.

This product offers advantages including ease of operation, absence of cytotoxicity, high cell recovery, and excellent cell viability.

Specifications

Storage

Store unopened at room temperature. Shelf life is 3 years.

Instructions for Use

• Loading of Separation Medium
  Under aseptic conditions in a clean bench, open the cell separation tube and place it on a tube rack. Add separation medium into each tube:

  • 15 mL tube: 4–4.5 mL

  • 50 mL tube: 15–15.5 mL

    Tighten the cap securely.

• Centrifugation

  Centrifuge at 1000 × g for 1 minute at 20°C.

• Inspection

  Check whether the separation medium has completely passed through the sieve plate and filled the lower chamber. If not, add additional medium and repeat centrifugation.

• Sample Loading

  Add anticoagulated whole blood directly without dilution:

  • 15 mL tube: 3–6 mL

  • 50 mL tube: 15–25 mL

• Centrifugation

  Centrifuge at 800 × g for 15 minutes at 20°C.

  Use low acceleration and deceleration settings (e.g., gear 3 if 10 levels are available).

• PBMC Collection

  After centrifugation, the PBMC layer is located between the plasma and the separation medium. Carefully aspirate the PBMC layer.

• Washing Step

  Wash the collected cells 1–2 times using RPMI-1640 medium (20°C, 250 × g, 10 min). Resuspend lymphocytes in 0.9% (w/v) saline or appropriate culture medium for downstream use.

Applicable Instruments

Horizontal rotor centrifuge.

Sample Requirements

The sample must be fresh anticoagulated blood collected under sterile conditions. Freezing or refrigeration during storage, handling, or transportation must be avoided.

Interpretation of Results

Variations in centrifuge performance and environmental temperature conditions may affect separation efficiency. Users may optimize centrifugation speed and time to achieve optimal results according to their laboratory conditions.

Limitations of the Method

The procedure requires that the temperature of the sample, separation medium, and environment be maintained at (20 ± 5)°C under normal atmospheric conditions

Precautions

• If the separation tube is opened but not immediately filled, ensure the cap is tightly closed to prevent contamination.

• After filling the separation medium and centrifugation, if not used immediately, keep the tube upright, protected from light at room temperature, and use within 3 months.

• The optimal separation temperature is (20 ± 5)°C. Deviation may affect performance.

• Blood should be collected and processed at room temperature, stored in an anticoagulant container for no more than 2 hours, and should not be refrigerated at 4°C.

• Whole blood can be used directly without dilution; dilution does not significantly affect separation performance.

  Recommended sample volumes:

  • 15 mL tube: ≥3 mL, recommended ≤6 mL

  • 50 mL tube: ≥15 mL, recommended ≤25 mL

    Insufficient sample volume may compromise separation efficiency.

• Blood cells separated from plasma may be used directly or diluted (1:1) with calcium- and magnesium-free PBS, saline, or culture medium before loading into the tube, provided sufficient erythrocytes are present.

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