ExerciseⅦ Lymphocyte Proliferation Assay
A commonly used method to measure cellular immune function analyzes in vitro lymphocyte proliferation.The substances which stimulate lymphocyte proliferation are divided into two categories：（1）nonspecific stimulators or mitogens，such as PHA，or Con A；（2）specific stimulators or antigens，such as the purified protein derivative（PPD）from M.tuberculosis.Among these stimulators，PHA is widely used.The receptor for PHA is expressed only on the surface of T lymphocytes.The extent to which PHA stimulates T lymphocytes to proliferate is compared to the proliferation stimulated by a specific antigen.The common methods for detecting lymphocyte proliferation in vitro include morphologic counting，3 H-thymidine incorporation.Morphologic counting is simple，but it is affected by subjective factor.3 H-thymidine incorporation has higher sensitivity and accuracy，though having radioactive contamination，it is still a generally acknowlledged method for us.
When T lymphocytes are incubated with PHA in vitro，they are stimulated to increase the synthesis of nucleic acid and protein.At the same time，the morphology of cells is transformed to a lymphoblast.Thus，this test is also called lymphocyte transformation test.
Principle
Peripheral blood or separated lymphocytes are incubated with PHA in vitro for 72 h.The cells are smeared onto a slide and stained.Under the microscope large naive lymphocytes can be observed.Only T lymphocytes express the receptor for PHA，so PHA can only stimulate T lymphocytes.The percent of T cells that transform is calculated by counting 100～200 lymphocytes.This is correlated with the cellular immune function，so it is used as one of the guidelines for cellular immunity function assays.
Materials
1.PHA：final concentration is 50～200μg/ml.
2.RPMI 1640 culture medium：Add newborn calf serum（NCS）to a final concentration of 20%.Add 100μl/ml penicillin and 100μg/ml streptomycin.
3.Wright staining solution.
4.Incubator，centrifuge，microscope，96 well culture plate etc.
Methods
1.According to experiment 1，separate peripheral blood mononuclear cells and adjust cell concentration to 2×106/ml with RPMI 1640 culture medium.
2.Add each sample to a set of 6 wells.Pipette 100μl of the cell suspension into each well.
3.Add 50μl PHA（final concentration 100μg/ml）to three wells.Leave 3 wells with no PHA.Then add RPMI 1640 culture medium so that there is 200 μl/well.Place it in a 37℃incubator for 72 h.
4.Remove the plate，draw a drop of the cell suspension to smear on a slide.After drying，stain with the Wright stain solution for 1 to 2 min.Wash，dry and observe under a microscope.
Results
Observe the results noticing the sizes and morphology of the cells.During the transformation the cells that are commonly seen are：lymphoblasts，transitional lymphocytes，cells in which the nucleus is dividing，mature lymphocytes，etc.Mature lymphocyte：Its diameter is 6 to 8μm with the chromatin close together.The nucleolus is invisible and there is little cytoplasm.（Figure 1）(https://www.daowen.com)
Lymphoblast Its volume expands to be 3 to 4 times that of the immature lymphocytes.The chromatin is loose and shows a thin net.The membrane of the nucleus is fair.There are 1 to 4 lobes of the nucleus.The cytoplasm is rich，with projecting pseudopodiumed and sometimes there are vacuoles present.（Figure 2 and Figure 3）
Transitional lymphocyte Its volume has expanded.The chromatin is loose with or without a nucleolus.The cytoplasm is increasing.（Figure 4）
Cells in which the nucleus is dividing：Its nucleus is in mitosis.Chromosomes are often visible.
Transformed cells include lymphoblasts，transitional lymphocytes，and cells in which the nucleus is dividing.
Two hundred viable lymphocytes are counted and the percentage of lymphocyte that are undergoing transformation is calculated.
Normal value The percentage of lymphocyte transformation in human peripheral blood is 60%to 80%.
Figure 1 Mature lymphocyte
Figure 2 Lymphoblast
Figure 3 Lymphoblas
Figure4Transitonallymphocyte