Lab 15.   Lymphocyte Proliferation Assay

            Lymphocyte proliferation assay is used to determine lymphocyte activation and the cell-mediated immune responses.  When B cells encounter their specific antigens, with the help of T cells, B cells are stimulated to undergo proliferation.  When T cells are activated by antigen-presenting cells and cytokines, T cells undergo proliferation.   The proliferation of B and T cells leads to clonal expansion and the initiation of the specific immune responses.

            Cells undergoing proliferation increase their rate of protein and DNA synthesis.  The increase in DNA synthesis can be measured by adding [³H]thymidine, a radioisotope-labeled DNA precursor, to the cell culture medium.  The amount of tritium taken up by the dividing cells is correlated to the level of cellular proliferation.  Cells undergoing proliferation are also metabolically active and increase their cellular level of dehydrogenase and their product NADH and NADPH.  The levels of NADH and NADPH can be measured by their ability to reduce yellow colored MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) to intracellular purple formazan.  The resulting purple products can be solubilized and quantified by spectrophotometric means.  MTT and [³H]thymidine incorporation are two common methods used to measure cell proliferation.

            Cytokines, such as IL-2, can stimulate T cells to proliferate.  T cells proliferate in an IL-2 concentration-dependent manner.  The level of T cell proliferation can be used as a measurement of IL-2 concentration.  Using IL-2 with a known concentration as a standard, the concentration of IL-2 in an unknown sample can be measured by its ability to stimulate T cell proliferation

            In this lab exercise, you will carry out an MTT cell proliferation assay to analyze cytokine-stimulated T cell proliferation.
 

Procedure

April 26

 To protect cells from contamination, we will carry out this lab exercise under sterile conditions.  In order to keep cells sterile, you should follow these instructions:

a.  Do not open plates or tubes containing cells and medium or boxes containing sterilized tips to open air.  The plates, tubes, pipette tips, and pipettes should only be opened in a laminar flow hood.

b.  Use 70% ethanol to wipe the surface of the laminar flow hood, your pipetteman and your hands before you start to work in the hood.

c.  When you carry out your exercise in a hood, plan ahead for each step.  For example, if you want to transfer some medium from a tube to a plate, you should open the cap of the tube before you pick up a pipette.

d.  During the exercise, do not let your cells come in contact with anything that is not sterile, such as a pipette taken outside the hood or an end of a pipette that has come in contact with your finger or the surface of the hood or the outside of a medium bottle.

1.  You have the following things in the hood:

 T cells:  CTLL
 T-stim:  IL-2 + ConA

2.  Add 100 ml of 5% CM to each well of the first three columns of the 96-well plate except for the A1, A2 and A3 wells.

3.  Add 200 ml of T-stim (containing IL-2 as positive controls) each to the A1, A2 and A3 wells.  Then make two-fold dilutions of each of the wells B1 through G1, B2 through G2 and B3 through G3.  To do this, transfer 100 ml from A1 to B1.  Mix and transfer 100 ml from B1 to C1, and so on.  When you reach the well G1, discard the 100 ml taken from G7 and leave H7 as the blank control.  Do the same to the columns 2 and 3.

4.  Add 50 ml CTLL cells (3 x 10⁵/ml) to each well of columns 1, 2, and 3.

5.  Incubate the plate in a CO₂ incubator at 37^oC for 44 hours.
 

April 28

Each group needs to send a person to the lab at 10 am, April 24 (4 hours before the lab starts).  Please carry out the following steps in a laminar flow hood.

6.  Add 10 ml of MTT into each well of columns 1, 2, and 3.

7.  The plate will be incubated in a CO₂ incubator at 37^oC for 4 hours.

Because we will not culture the cells further, the rest of this exercise will be carried out under non-sterile conditions, on your bench.

8.  Add 100 ml of detergent to each well of columns 1, 2, and 3.  Swirl gently.  Allow the plate to site at room temperature in the dark for 2 h to dissolve the purple crystals.

9.  During the incubation, look at the cells under an inverted microscope.  The intracellular punctate purple precipitate should be visible.

10.  Read the optical density on the plate reader at 570 nm.

11.  Calculate the mean and standard deviation for each set of triplicates, and make a plot of the optical density vs. the dilution factors of T-stim (IL-2).
 

Reading
Immunobiology A31

Study Questions

1. In this lab exercises, you carried out the procedure in a sterile hood.  Please point out which practices below are correct and which are wrong.  If a practice is wrong, please make corrections.

a. Before you go into the sterile hood, you wash your hands using tap water.

b. Since you are among the first group of students to use the sterile hood in a lab period, you do not have to clean the surface of the hood.

c. The tip of your pipette accidentally touches the surface of the hood.  Since I already cleaned the surface with alcohol, it will be fine to keep using the pipette.

2. Please list two methods for the analysis of cell proliferation.  Explain the principles of each of the methods.

3. Propose two different methods for determining the concentration of IL-2.  Please list names of the methods and key reagents for each method.