You harvested spleens cells from mice infected 6 weeks earli

You harvested spleens cells from mice infected 6 weeks earlier with a large number of Leishmania majorparasites. You want to find out the characteristics of the immune response generated in these mice by employing an ELISPOT assay. In addition, you employed Magnetic Cell Sorting technology to characterize the spleen population producing cytokines. You plated 7.5 X 10⁵ spleen cells/well in the microtiter plate, representing 1/130 of the spleen,

Upon counting SPOTS the following results were obtained:

                                                   No. of IFNg Spots/well       No. of IL4 Spots/well

Gr. 1 Unsorted control                          120                                     10

Gr. 2 CD4+ population                         105                                      4

Gr. 3 CD8+ population                             7                                       0

Calculate the number of IFNg and IL4 producing cells /spleen

Solution

ELISpot assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or polyclonal antibody specific for the chosen analyte is pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO₂ incubator for a specified period of time. During this incubation period, the immobilized antibody, in the immediate vicinity of the secreting cells, binds secreted analyte. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for the chosen analyte is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual analyte-secreting cell. The spots can be counted with an automated ELISpot reader system or manually, using a stereomicroscope.

* Each coloured spot represents a cytokine secreting cell.

Calculation:

Total IFN-gamma spots / well = Number of IFN-gamma spots / well by CD4⁺ cells +  Number of IFN-g spots / well by CD8⁺ cells + unsorted control cells (cells other than CD4⁺ and CD8⁺)

= 105 + 7 + 120 = 232

Assuming that, each individual spot representing an individual analyte-secreting cell, we got 232 splenocytes /well that produce IFN-gamma.

So also similarly,

Total IL4 spots / well = 10 + 4 + 0 = 14 per well

It has given in the question that, 7.5 X 105 spleen cells/well in the microtiter plate, representing 1/130 of the spleen.

Therefore, total number of IFN-gamma producing cells / spleen = 232 X 130 = 30160

and total number of IL4 producing cells / spleen = 14 X 130 = 1820