Peripheral blood mononuclear cells (PBMCs) refer to mononuclear cells in peripheral blood, including lymphocytes, monocytes, dendritic cells, and other minor cell populations (e.g., hematopoietic stem cells). PBMCs are the most commonly used cellular model for immunological function studies, such as cell proliferation, cytotoxicity, and cytokine secretion assays.
Mouse peripheral blood lymphocyte separation medium is primarily composed of Ficoll and sodium diatrizoate. It is a sterile density gradient separation medium with low endotoxin levels, specifically designed for isolating mouse peripheral blood lymphocytes. It is suitable for separating target cells from mouse blood and tissue homogenates. The separation principle is based on the density differences among blood cells (the optimal density is 1.080 for isolating mouse mononuclear cells; 1.084-1.087 for rat mononuclear cells; the density of red blood cells and granulocytes is approximately 1.092 g/mL; and the density of platelets ranges from 1.030 to 1.035 g/mL). Through centrifugation, cells of specific densities distribute according to the corresponding density gradient, thereby separating lymphocytes from mouse peripheral blood or tissues.
Product Performance Specifications
Appearance: Opalescent or slightly opalescent aqueous injection solution
Density: 1.081±0.001 g/mL
Osmolality: 280-340 mOsmol/kg
Sterility: Filtered through a 0.22 μm membrane
This product is for research use only and is not intended for clinical diagnosis or any other purposes.
Operating Procedures (For Reference Only)
1. Dilute the blood sample: Mix 1 volume of isotonic solution (e.g., PBS buffer) with 1-2 volumes of blood, resulting in a ratio of isotonic solution: anticoagulated blood=1:1-2.
2. To meet practical experimental needs, the recommended operational protocols based on different blood sample volumes are as follows:
Scenario A: Blood sample volume<2 mL
1. Take a 15 mL centrifuge tube and add 4 mL of separation medium.
2. Carefully pipette the diluted blood sample and layer it gently onto the surface of the separation medium. Centrifuge at 450-500×g for 20-30 min. (Note: Centrifugation parameters depend on the blood sample volume. A larger sample volume requires higher centrifugal force and longer centrifugation time. Users should optimize these parameters to achieve the best separation efficiency.)
3. After centrifugation, the contents in the centrifuge tube are separated into four distinct layers from top to bottom:
Layer 1: Plasma layer
Layer 2: Opaque milky white lymphocyte layer
Layer 3: Transparent separation medium layer
Layer 4: Red blood cell layer
(As illustrated in the figure below)
4. Carefully aspirate the second layer (opaque milky white lymphocyte layer) into a new 15 mL centrifuge tube. Add 10 mL of isotonic solution to the tube and resuspend the cells thoroughly.
5. Centrifuge at 250×g for 10 min.
6. Discard the supernatant.
7. Resuspend the collected cells with 5 mL of isotonic solution using a pipette.
8. Centrifuge at 250×g for 10 min.
9. Repeat steps 6, 7, and 8. After discarding the final supernatant, resuspend the cells in 1 mL of the appropriate medium required for subsequent experiments.
Scenario B: Blood sample volume=2-4 mL
1. Take a 15 mL centrifuge tube and add 5 mL of separation medium.
2. Carefully pipette the diluted blood sample and layer it gently onto the surface of the separation medium. Centrifuge at 450-650×g for 20-30 min. (Note: Centrifugation parameters depend on the blood sample volume. A larger sample volume requires higher centrifugal force and longer centrifugation time. Users should optimize these parameters to achieve the best separation efficiency. The maximum centrifugal force is recommended not to exceed 1200×g.)
3. After centrifugation, the contents in the centrifuge tube are separated into four distinct layers from top to bottom:
Layer 1: Plasma layer
Layer 2: Opaque milky white lymphocyte layer
Layer 3: Transparent separation medium layer
Layer 4: Red blood cell layer
4. Carefully aspirate the second layer (opaque milky white lymphocyte layer) into a new 15 mL centrifuge tube. Add 10 mL of isotonic solution to the tube and resuspend the cells thoroughly.
5. Centrifuge at 250×g for 10 min.
6. Discard the supernatant.
7. Resuspend the collected cells with 5 mL of isotonic solution using a pipette.
8. Centrifuge at 250×g for 10 min.
9. Repeat steps 6, 7, and 8. After discarding the final supernatant, resuspend the cells in 1 mL of the appropriate medium required for subsequent experiments.
Precautions
1. Invert and mix well before opening. This separation medium is a sterile product and is susceptible to bacterial contamination; therefore, all operations must be performed under sterile conditions. After opening under sterile conditions, store the product at 4°C, with a validity period of 6 months.
2. The separation medium should be maintained at room temperature (18°C-25°C) during use. If the ambient temperature is too low, pre-warm the medium appropriately. Centrifugation at 4°C or lower temperatures may increase red blood cell contamination in the buffy coat layer.
3. The blood sample should preferably be fresh anticoagulated blood (collected within 2 hours). To maintain lymphocyte viability, avoid freezing and refrigeration of the sample.
4. For blood dilution or cell washing, do not use buffers or culture media containing Ca²⁺ or Mg²⁺-these ions can cause blood cell agglutination, which significantly reduces cell yield and purity.
5. Some plastic materials (e.g., polystyrene) may cause cell adhesion due to electrostatic effects, thereby affecting separation efficiency.
6. Variations in blood sample viscosity or temperature may impact separation results. Adjust centrifugation speed and duration to optimize separation conditions.
7. Aspirating an excessive amount of the lymphocyte layer or separation medium layer may lead to the inclusion of granulocytes at the interface, increasing granulocyte contamination. Aspirating an excessive amount of the plasma layer may result in contamination of lymphocytes with plasma proteins and platelets.
8. If further culture of the isolated cells is required, ensure strict sterile operation during blood collection and separation processes to avoid microbial contamination.
9. Different animal blood samples exhibit varying cell dispersion coefficients and cell charge properties in separation media of different densities. When preparing custom separation media, users should specify the required medium density, animal species, and the name of the target cells to be isolated.
10. This product is light-sensitive and should be stored at room temperature in the dark.
11. For your safety and health, wear a lab coat and disposable gloves during operation.
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