Immunology Laboratory: Plaque Forming Cell response

The McGill Physiology Virtual Lab

Immunology Laboratory

Plaque Forming Cell Response
(Not performed during the lab)

Two of the immune function assays, relying on antibody response, are the Plaque Forming Cell Assay (PFC) and the hemagglutination test. The PFC assay measures IgM producing cells and:

is an indicator of cell proliferation
identifies the primary and secondary lymphoid organs

Background

In the mid sixties, Jerne developed a plaque assay, based on local hemolysis in gel, to identify and count individual antibody forming cells. The original technique has been modified in different laboratories. The technique used in the teaching laboratory is a modification of the method described by Cunningham and Szenberg (1968): it is the "direct plaque forming cell (PFC) assay and only detects IgM secreting cells. An " indirect PFC assay" can be used to detect IgG secreting cells.

References: Jerne, N.K. and Nordin, A.A. 1963. Science 140: 405.
Cunningham, A.J. and Szenberg, A. 1968. Immunology 14: 599.
Kongshavn, P.A.L. and Lapp, W.S 1972. Immunology 22: 227

Immunized animal versus non-immunized animal

Procedure

Preparation of a cell suspension:

Spleen cell suspensions are prepared by gently tamping the spleen through a 60-mesh stainless steel screen, and collecting the cells in balanced salt solution (BSS). The spleen cells are washed and made up to 15 ml with BSS. SRBC are washed twice and made up to a 10% concentration. Complement (Gibco) is diluted 1/20 with BSS. All stock solutions are kept on ice water until used.

The excised mouse spleen is placed on a 60-mesh stainless steel screen, over the bottom of a small empty Petri dish.

The spleen is gently teased through the mesh with a spatula

and rinsed with balanced salt solution.

All the cells are transferred to a conical test tube and the volume adjusted to 15 ml.

Washing cells:

The tube filled with the cell suspension is centrifuged. The supernatant is aspirated and discarded. The remaining pellet, constituted of intact spleen cells, is resuspended in fresh balanced salt solution. The tube is once more centrifuged; the supernatant is also discarded and the pellet is resuspended in more balanced salt solution. The cells are washed this way three times with balanced salt solution.

Adding cells, SRBC and Complement to slide chambers (performed in the lab):

The test consists of mixing 0.05 ml of spleen cells, 0.070 ml of SRBC and 0.5 ml of the complement solution in a test tube at 37C. The whole mixture is immediately withdrawn and put into chambers prepared by gluing two 75 x 25 mm slides together with double-sided tape. Four to five slide chambers are necessary for each sample, filling up the last chamber if necessary with a blank solution of SRBC and complement in the same concentrations as for the test mixture.

The slide chambers are sealed with paraffin wax, and incubated at 37C for 45-60 minutes. In earlier experiments, a 30 minute incubation period was used, but this proved insufficient time for full development of all plaques.

The number of PFC are counted by both macro- and microscopic examination.

Since time does not permit you to make your own cell suspension, you are provided with spleen cell suspensions as well as thymus cell suspensions from the two experimental groups. Remember to always mix the cell suspension as you use it: the cells settle down at the bottom of the tube rather quickly (you want to make sure you add a uniform cell suspension into each of the slide chambers).

All other reagents, made up to the proper concentrations, are labelled and placed at central locations in the laboratory or at your bench. Each laboratory team should assay 4 different experimental groups of mice.

In brief:

Place into a tube:

50 ml of a spleen from animal A, or from animal B, or thymus cell from animal A, or B

0.50 ml of C' into each tube

70 ml of SRBC (1/10 concentration) into each tube

Warm to 37C in the water bath (30-60 seconds)

Use a Pasteur pipette to transfer the contents of all tubes into the slide chambers (4-6)). Fill the last chamber with the blank solution (C' + SRBC) if necessary.

Seal the chambers by dipping both edges into the warm paraffin wax.

Place in the incubator for one hour.

Remove from the incubator and count.

Results

You should see on some of your slides clear areas where RBC lysis has occurred: Antibody-forming cells are measured by mixing the spleen cells from the immunized animal with the Antigen (Sheep Red Blood Cells). Following incubation, the red cells surrounding the cells secreting specific antibody become coated with the antibody and may be lysed by complement.

A slide chamber, showing clear areas (a light beam was directed onto the slide).

View under the microscope: 2 plaque-forming cells.

From your results, identify the two groups of animals (A or B?) - normal or immunized with SRBC. Assume the following information for each cell suspension:

Organ	Total lymphoid cell count	Total volume of cell suspension
Spleen	80 x 10⁶ cells	15 ml
Thymus	90 x 10⁶ cells	15 ml

Determine the total number of PFC per organ: there are 80 x 10⁶ lymphocytes of which 60% are B cells and the doubling time is 15- 18 hours.
Determine the number of PFC per 10⁶ lymphoid cells: you are estimating the number of clones that responded by manufacturing antibody.
Obtain results from other laboratory teams and calculate the: mean, standard deviation and standard error of the mean

Points of discussion

• The number of PFC in each organ/10⁶ cells

• The number of PFC in the non-immunized group

• How do you account for background PFC in the non-immunized group?

• Immunological specificity

• Organs active in antibody production

• Using the PFC assay how could you demonstrate that antibody forming cells are not T lymphocytes?