Protocol for the Separation of T and B Lymphocytes

Lymphocytes are divided into two major subgroups, T-lymphocytes and B-lymphocytes, which have different characteristics and functions. When performing certain immunological experiments, pure T and B lymphocytes need to be isolated first. This experiment describes the procedure of the T and B lymphocyte separation test.

• Preparation of AET solution. Weigh 402 mg of AET powder and dissolve it in 10 ml of distilled water to make a 0.143 M solution and adjust the pH to 9.0 with 4N NaOH solution.
• AET treatment of RRBC. Take the washed and compacted RRBC and add 4 parts of freshly prepared pH 9.0 AET solution to one part of compacted AET-RRBC and mix thoroughly in a 37°C water bath for 15 minutes, shaking every 5 minutes. Remove and add cold sterile saline to the mouth of the centrifuge tube (1-2 cm) and centrifuge at 1800 rpm for 5 min. Wash 3-5 times consecutively, and each wash must be shaken well to minimize AET-RRBC adhesion into clusters and to observe for hemolysis. If there is hemolysis, wash again with 199 medium containing calf serum, and finally formulate 10% AET-RRBC suspension and store at 4°C for no more than 5 days.
• Preparation of 1% AET-RRBC. The 10% concentration of AET-RRBC, which was pre-prepared and stored in a 4°C refrigerator, was diluted to 1% with 199 culture solution containing 10% calf serum.
• AET-E wreath assay. Isolated single nucleated cells (2 x 10⁶/mL) were mixed with an equal amount of 1% AET-RRBC and placed in a 37°C water bath for 15 minutes. Shake well every 5 minutes and divide into several tubes of 2-3 ml each. After centrifugation (1000 rpm) at low speed for 5 minutes, transfer to a 4°C refrigerator for 45 minutes.
• Separation of T and B lymphocytes. The cell suspension forming the E-wreath was then separated with lymphocyte layering solution, and the cloudy cells at the interface were aspirated, i.e., the B-lymphocyte-rich population. E-wreaths precipitated at the bottom of the tube were washed once with Hanks solution and then treated with 3 ml of double-distilled water for 3 min. Hypotonic lysis of RRBCs around the E-wreath, immediately adds 1 ml of 3.5% NaCl solution to reduce isotonicity and centrifuge the precipitate at low speed to obtain the T-rich lymphocyte population.

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The AET-RRBC solution must be prepared fresh and should not be stored for a long time.

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