Evaluation of Cellular Activity

by Peter Delves, PhD

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    00:01 In the nitroblue tetrazolium test (NBT), the pale yellow color of NBT changes to dark blue in neutrophils stimulated with phorbol myristate acetate (PMA) which is used to induce a reactive oxygen burst in these cells, producing reactive oxygen species.

    00:24 So a burst of respiration that normally occurs following phagocytosis of microorganisms.

    00:30 In a normal donor, we can see this dark blue precipitate that occurs over the neutrophils indicating that they have indeed mounted a respiratory burst producing reative oxygen species following this stimulation, artificial stimulation with PMA.

    00:50 In a patient with chronic granulomatous disease, which is an immunodeficiency in which patients are unable to produce reactive oxygen species, there is no reduction of the NBT.

    01:02 And therefore these dark patches do not appear over the neutrophils.

    01:08 The production of reactive oxygen species can also be measured using dihydrorhodamine 123.

    01:16 The T-cell receptor excision circle or TREC assay is used to analyze the output of T-cells from the thymus.

    01:28 TRECs are circular DNA molecules that are produced during T-cell receptor gene recombination.

    01:35 They can be measured using the polymerase chain reaction, PCR.

    01:42 And this assay is used to quantify recent thymic emigrants as a measure of T-cell output from the thymus.

    01:53 So here we have an example of the T-cell receptor α-chain gene locus.

    01:59 And as T-cells develop within the thymus they will recombine their T-cell receptor genes.

    02:06 And in the case of the T-cell receptor α-chain, this recombination will place a Vα gene segment adjacent to a Jα gene segment. And the intervening bit of DNA forms a circle, the T-cell receptor excision circle that we can see here.

    02:26 And the TREC assay measures the generation of these circles to give an indication of T-cell output from the thymus.

    02:40 Lymphocyte responses can be measured via the proliferation assay.

    02:46 In the case of T-cells, the substance phytohemagglutinin (PHA) acts as a mitogen.

    02:55 A mitogen is a substance that generates mitosis, causes cell division.

    03:02 So these cells will be polyclonally activated, that means they become activated irrespective of their particular antigen specificity and they will start to divide over and over and over again.

    03:15 By adding the isotope of thymidine, thritiated thymidine, this will become incorporated into the cells as they divide.

    03:26 It’ll be incorporated into their newly produced DNA.

    03:30 And one can measure the radioactivity in the 3H-thymidine.

    03:40 The ELISPOT assay which stands for ELISA SPOT assay is used to measure cytokine release or indeed can be used to measure the protein-- the level of protein released from different cell types.

    03:55 So it could be a hormone for example.

    03:57 But here we’re looking at a cytokine release.

    04:00 Peripheral blood cells which consists of lymphocytes and monocytes are added to a dish that is coated with a anti-cytokine antibody.

    04:14 These cells can release cytokines and particularly the lymphocytes in response to incubation with soluble antigen, will be stimulated to release cytokines.

    04:27 After culturing the cells with antigen for two days, we can see in this particular assay that Th2 cells are secreting IL-4. Interleukin-4 is a characteristic cytokine of Th2 cells. And they are secreting this in response to the soluble test antigen. The IL-4 released from these cells is captured by the antibody that is coating the plate. So following the incubation, the release of the cytokine, the cytokine being bound by the antibody, captured by the antibody, the cells are then washed away.

    05:11 And the cytokine is revealed by a second antibody, which is a labeled antibody against another epitope on the same cytokine.

    05:22 A chemical reaction gives spots as we can see here.

    05:27 And you can see on the left hand side, a example of cells that are not secreting interleukin-4.

    05:35 And on the right, some spots showing that some of the cells have secreted interleukin-4.

    05:43 And one is able to enumerate these, if you know how many cells you added to the assay, then you can get an approximation of the percentage of cells that are secreting this particular cytokine, the ELISPOT assay.

    05:55 It’s also possible to measure cytotoxic T-cell activity.

    06:00 In this assay, cells are labeled with chromium-51.

    06:09 And these are mixed with effector cells that are able to mediate cytotoxicity.

    06:15 So these could be natural killer cells for example, of the innate response, or they could as shown here be the cytotoxic T-lymphocytes of the adaptive immune response.

    06:24 If the labeled cells are capable of being killed by the cytotoxic T-lymphocytes, there will be release of radioactivity into the culture fluid from the cells as they become killed.

    06:38 So as the target cells are killed, they release the radioactive isotope.

    06:43 And this, the amount released will be proportional to target cell lysis.

    About the Lecture

    The lecture Evaluation of Cellular Activity by Peter Delves, PhD is from the course Immunodiagnostics. It contains the following chapters:

    • Nitroblue Tetrazolium Test (NBT)
    • T-Cell Receptor Excision Circle (TREC) Assay
    • Lymphocyte Proliferation Assay
    • ELISPOT Assay to Measure Cytokine Release
    • Measuring Cytotoxic T-Cell Activity

    Included Quiz Questions

    1. Negative due to lack of ability in producing reactive oxygen species
    2. Positive due to lack of ability in producing reactive oxygen species
    3. Negative due to the ability in producing reactive oxygen species
    4. Positive due to the ability in producing reactive oxygen species
    5. Negative due to excessive production of reactive oxygen species
    1. Neutrophil stimulation
    2. Neutrophil prevention
    3. Acting as a dye
    4. Formation of reactive oxygen species
    5. Preventing formation of reactive oxygen species
    1. T-cell lymphopenia
    2. Chronic granulomatous disease
    3. Chronic lymphocytic leukemia
    4. Acute lymphoblastic leukemia
    5. Chronic myeloid leukemia
    1. Enzyme-linked immune absorbent spot assay
    2. T-cell receptor excision circles assay
    3. Nitroblue tetrazolium assay
    4. Lymphocyte proliferation assay
    5. Cytotoxic T-cell activity measurement assay

    Author of lecture Evaluation of Cellular Activity

     Peter Delves, PhD

    Peter Delves, PhD

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