Human Lymphocyte Separation Medium, IC-1189
Product Introduction
This product is a sterile, ready-to-use density gradient medium specifically formulated for the isolation of human lymphocytes from peripheral blood, cord blood, or bone marrow by density gradient centrifugation. Lymphocyte separation is a fundamental technique in immunology, hematology, and clinical research, enabling the purification of peripheral blood mononuclear cells (PBMCs) including lymphocytes and monocytes from whole blood while depleting erythrocytes, granulocytes, and dead cells. This separation medium consists of a polysaccharide-based solution with precisely adjusted density and osmolarity. During centrifugation, the medium forms a density barrier: PBMCs are retained at the interface between the separation medium and the overlying plasma layer, while erythrocytes and granulocytes, having higher density, sediment to the bottom of the tube. The isolated PBMCs retain high viability and functional integrity, making them suitable for downstream applications including flow cytometry, cell culture, ELISpot assays, T cell activation and proliferation assays, and immunological phenotyping.
Product Features
1. Optimized Density and Osmolarity: Precisely formulated to achieve a density of 1.077 g/mL, optimal for human PBMC isolation with high purity and yield.
2. Ready-to-Use Sterile Solution: Supplied as a sterile, endotoxin-tested solution requiring no dilution or additional preparation.
3. High Lymphocyte Yield and Purity: Efficiently separates PBMCs while depleting erythrocytes and granulocytes, with typical lymphocyte purity of >90% and viability >95%.
4. Rapid and Simple Protocol: Single-step density gradient centrifugation provides PBMCs within 30-45 minutes from whole blood.
5. Versatile Downstream Compatibility: Isolated PBMCs are suitable for flow cytometry, cell culture, immunophenotyping, ELISpot, proliferation assays, and cryopreservation.
Specifications
Size: 100 mL
Density: 1.077 ¡À 0.001 g/mL
Osmolality: 280-310 mOsm/kg
Sterility: Sterile
Endotoxin: ¡Ü0.5 EU/mL
Storage and Stability
Storage Conditions: Store at 4¡ãC to 8¡ãC, protected from light. Do not freeze. Allow the medium to warm to room temperature before use.
Shelf Life: The product is stable for 24 months from the date of manufacture when stored as directed. After opening, use within 3 months when stored at 4¡ãC and handled aseptically.
Protocol (For Reference Only)
Important: Use aseptic technique throughout the procedure. Allow the separation medium to warm to room temperature (18-25¡ãC) before use for optimal density and separation performance. All centrifugation steps should be performed at room temperature with the brake off to preserve the gradient interface.
1. Blood Sample Preparation: Collect peripheral blood in anticoagulant tubes (EDTA, heparin, or citrate). Dilute whole blood with an equal volume of sterile PBS or RPMI-1640 medium without serum and mix gently.
2. Gradient Setup: Add an appropriate volume of Human Lymphocyte Separation Medium to a 15 mL or 50 mL conical centrifuge tube. Carefully layer the diluted blood over the separation medium at a ratio of approximately 2:1 (diluted blood:separation medium) without disturbing the interface. For a 15 mL tube, use 3 mL of separation medium and carefully overlay 6-8 mL of diluted blood.
3. Density Gradient Centrifugation: Centrifuge at 400-500 x g for 20-30 minutes at room temperature with the brake off. After centrifugation, distinct layers will be visible: plasma (top), a thin white band of PBMCs at the interface, clear separation medium, and a red pellet of erythrocytes and granulocytes (bottom).
4. PBMC Collection: Using a sterile pipette, carefully aspirate the PBMC layer from the plasma/separation medium interface and transfer to a clean 15 mL or 50 mL conical tube. Avoid aspirating the separation medium to minimize granulocyte contamination.
5. Cell Washing: Add 3-5 volumes of sterile PBS or RPMI-1640 to the collected PBMCs, mix gently, and centrifuge at 300 x g for 10 minutes at room temperature. Discard the supernatant, resuspend the cell pellet in fresh medium or buffer, and repeat the wash step once or twice to remove residual separation medium and platelets. Resuspend the final PBMC pellet in appropriate medium or buffer for downstream applications.
Precautions
1. Allow the separation medium to reach room temperature before use; cold medium has altered density that reduces separation efficiency.
2. Layer the diluted blood carefully over the separation medium without disrupting the interface; a sharp interface is critical for optimal PBMC recovery.
3. Centrifugation with the brake off is essential to preserve the PBMC band at the interface; braking may disrupt the gradient.
4. Minimize platelet contamination by washing cells at least twice; residual platelets can interfere with downstream functional assays.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What is the expected PBMC yield from human peripheral blood?
A1: Typical PBMC yield ranges from 0.5-2 ¡Á 10⁶ cells per mL of whole blood, depending on donor age, health status, and individual variation. Processing 10 mL of healthy donor blood typically yields 10-20 ¡Á 10⁶ PBMCs with >90% purity and >95% viability.
Q2: Can I use this medium for cord blood or bone marrow samples?
A2: Yes. This medium is suitable for isolation of mononuclear cells from cord blood and bone marrow. For cord blood, dilute with PBS at a ratio of 1:1 to 1:2 before layering. For bone marrow, filter through a 70 µm strainer to remove bone fragments before processing.
Q3: Why must the centrifuge brake be turned off?
A3: The brake causes rapid deceleration that generates turbulence, disrupting the delicate PBMC band at the density interface. Turning off the brake allows the rotor to coast to a gradual stop, preserving the gradient layers and maximizing PBMC recovery.
Q4: Can isolated PBMCs be cryopreserved?
A4: Yes. Isolated PBMCs can be cryopreserved in fetal bovine serum containing 10% DMSO or in commercially available cryopreservation medium. Cryopreserved cells should be stored in liquid nitrogen and thawed according to established protocols.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research and donor variability, optimization of processing conditions may be required for specific applications.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product and human biological samples.
Ordering Information
Catalog Number: IC-1189
Product Name: Human Lymphocyte Separation Medium
Size: 100 mL
Price: CNY ¥800.00 / USD $80.00 / EUR €96.00 / JPY ¥14400.00
2023 Version
