Isolation of single nucleated cells from peripheral and umbilical cord blood

Summary

Peripheral blood mononuclear cells can be used in: (1) immunology (including autoimmune diseases), infectious diseases, hematologic malignancies, vaccine development, transplantation immunology; (2) predicting the immunosuppressive effects of drugs and xenobiotics; (3) high-throughput screening This experiment is from the "Compact Guide to Immunology Experiments" by J.E. Colligan et al.

Operation method

Ficoll-Hypaque gradient centrifugation method

Principle

FicoH-Hypaque (also known as Lymphocyte Layering Solution) Gradient Centrifugation is a single density gradient centrifugal separation method. FicoH-Hypaque layering solution was used for the experiment. The sucrose polymer, called polysucrose (trade name Ficoll), has a molecular weight of 40 kD and is characterized by high density, low osmotic pressure, and non-toxicity. The high concentration of Ficoll solution has high viscosity, which is easy to make cell aggregation. Using the low concentration of Ficoll solution of 60g/L and adding urografin (commonly used in foreign countries as Isopaque or Hypaque, so it is also known as Ficoll-Hypaque layering solution), it can be prepared into a layering solution with suitable density. Because the density of human erythrocytes is 1.093, the density of granulocytes is 1.092, and the density of single nucleated cells is between 1.076 and 1.090, it is optimal to use the layering solution with a density of 1.077±0.001. Various blood components will repolymerize according to the density gradient during separation. Plasma and platelets are suspended in the upper part of the layered solution because of their lower density, while red blood cells and granulocytes have a higher density and sink to the bottom of the layered solution. Peripheral blood mononuclear cells (PBMC) have a slightly lower density than the layered fluid, so they are located at the interface of the layered fluid, so that PBMC can be obtained.

Materials and Instruments

Heparin anticoagulated or heparinized cord blood
PBS Poly sucrose-pantethine glucosamine solution HBSS Phosphate buffered salt solution Lymphocyte layering solution
Graduated centrifuge tubes Suction tubes Test tubes Capillary pipettes Rubber nipples Slides Coverslips Horizontal centrifuge

Move

1. 2 ml of blood was taken intravenously and added to a test tube containing heparin solution (10 - 50 u/ml blood sample) and mixed well to anticoagulate the blood. The anticoagulated blood was diluted twice with Hanks' solution of PH 7.2.

2. Aspirate 2 ml of lymphocyte layering solution into a graduated centrifuge tube, tilt the tube at an angle of 45°, and use a capillary burette to slowly add the diluted whole blood along the wall of the tube to the top of the separating solution, and care should be taken to keep the liquid surface clear.

3. Centrifuge at 18℃ - 20℃ for 20min with a horizontal centrifuge at 2000r/min.

4. Gently insert a capillary pipette into the turbid zone, gently aspirate the cells of the single nucleated cell layer along the wall of the tube, and transfer into another centrifuge tube. Avoid aspirating too much layered fluid or plasma.

5. Wash the cells with Hanks solution 3 times, the first time 2000r/min, for 10min. the second - third time 1500r/min, for 10min. can remove most of the mixed platelets.

6. Suspend the precipitated cells in culture medium and set aside.

Caveat

1. The glassware used for the experiment should be clean. If the prepared single nucleated cell suspension is used for cell culture, all the above operations should be carried out under sterile conditions, and the equipment and reagents used should be sterile.

2. The cell acquisition rate in the experiment is related to the room temperature and the specific gravity of the layered solution, etc. The layered solution should be stored at 4℃ away from light.

3. When adding diluted whole blood into the lymphocyte layering solution, the blood should not be washed into the layering solution, and the clear interface between the two layers of liquid should be maintained.

Common Problems

1. The purity of PBMC isolated by this method can be up to 95%, and the lymphocytes account for about 90%, of which T lymphocytes account for 80% and B lymphocytes account for 4%~10%.
   
   

2. This experiment is from "Compendium Immunology Laboratory Guide" Author: J.E. Colligan et al, Translator: Xuedao Cao et al.

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