Lymphocytes are divided into two main subpopulations, T and B lymphocytes, which have different properties and functions and are used for (1) immunological studies (2) identification of lymphocytes.
Operation method
Separation of T and B lymphocytes
Principle
After the lymphocytes were mixed with sheep red blood cells (SRBC) treated with bromoflower diaminoisothiocyanate (AET for short), all of the T-lymphocytes therein could adsorb the AET-SRBC and form a firm, stable and huge E-wreath, which had a higher percentage of the E-wreaths formed than that formed by the normal untreated SRBCs, and the formation was rapid, not easy to be dislodged, and had good reproducibility. When separated by the lymphocyte layering solution, the AET-E wreaths were easy to sink to the bottom of the tube, while the T lymphocytes that did not form E-wreaths could be obtained as pure T lymphocytes by detoxifying the AET-SRBCs around the wreaths with hypotonic solution, while the B lymphocytes could be taken directly from the interface of the layering solution.
Materials and Instruments
Fresh guinea pig blood Move 1. AET-RRBC Preparation Caveat 1. The process of isolating B cells is long, in order to avoid the natural death of B cells, 1640 can be used in the final rinsing of B cells, and 5-10% calf serum should be added. 2. Do not aspirate the liquid from the bottom of the test tube during each wash, i.e., to prevent the pressure-accumulated B cells from coming into contact with the air. 3. Perform a micro B-cell cytotoxicity assay using anti-B-lymphocyte serum with the examined cells according to the NIH method, and read the results using an inverted phase contrast microscope. Common Problems About isolation from mouse spleen: 1.Spleen tissue contains relatively little connective tissue, so it can be ground directly on a sieve without clipping or concern for blood vessels. 2. ficoll contains polysaccharides, so many people like to store it at 4 degrees, whether or not the room temperature does not have much effect on the experiment Spleen isolation of lymphocytes experiment is relatively simple, so just use ordinary lymphocyte isolation solution. 3 . Centrifugation 2000rpm × 20min, this step is very critical, and is to add cell suspension must be walled slowly added, do not destroy the interface, the separation of liquid and cell suspension I commonly used 1:2, and is the choice of centrifugal tube thickness will also affect the stratification. 4. The suspended white ring-shaped cell layer in the center is the desired lymphocyte layer. 5. The main purpose of cell washing is to wash away the separating solution, so it can't be omitted. For more product details, please visit Aladdin Scientific website.
Hanks' solution, calf serum, 199 culture fluid, physiological saline, sodium chloride solution.
Centrifuge tubes Centrifuge
(1) Preparation of AET solutionWeigh 402 mg of AET powder and dissolve in 10 ml of distilled water to make a 0.143 M solution and adjust the pH 9.0 with 4N NaOH solution.The solution must be freshly prepared and should not be stored for a long time.
(2) AET treatment of RRBCTake a good washing of the deposition of RRBC, according to a deposition of AET— RRBC add 4 freshly prepared pH9.0 AET solution fully mixed in a 37 ℃ water bath for 15 minutes, every 5 minutes shaking. Remove and add cold sterile saline to the mouth of the centrifuge tube (1-2 cm) and centrifuge at 1800 rpm for 5 minutes. Wash 3-5 times consecutively, and each wash must be shaken well to reduce AET-RRBC adhesion into clusters and to observe any hemolysis. If there is hemolysis, wash again with 199 medium containing calf serum, and finally formulate 10% AET-RRBC suspension and store at 4℃ for no more than 5 days.
(3) Preparation of 1% AET-RRBC:The 10% concentration of AET-RRBC, which was pre-prepared and stored in the refrigerator at 4°C, was diluted to 1% with 199 culture medium containing 10% calf serum.
2. Isolation of single nucleated cells from fresh guinea pig blood was performed as described in the post-test.
3. AET—E wreath test:Isolated single nucleated cells ( 2x106/ml ) were mixed with an equal amount of 1% AET-RRBC, placed in a 37°C water bath for 15 minutes, shaken every 5 minutes, divided into several tubes of 2—3 ml each, centrifuged at low speed (1000 rpm) for 5 minutes, and then transferred to a 4°C refrigerator for 45 minutes.
4. Isolation of T and B lymphocytesThe cell suspension forming the E-wreath was then separated with lymphocyte layering solution, and the cloudy cells at the interface were aspirated, which is the B-lymphocyte-rich population. The E-wreath precipitated at the bottom of the tube was 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 was carried out, and 1 ml of 3.5% NaCl solution was added immediately to make the reduction isotonic, and centrifugation of the precipitate at a low speed was carried out to obtain the T-lymphocyte-rich population.