Analysis of Inflammatory Cytokine using Flow Cytometry, q-PCR and Elisa Test on Mice Immune System

MEDICAL IMAGING AND DRUG
DELIVERY
Analysis of inflammatory cytokine using Flow cytometry,
q- PCR and Elisa test on mice immune system

Table of contents
1. Abstract
2. Introduction
3. Materials and Methods
4. Results
5. Discussions
6. Conclusions
7. References

Abstract
In biomedical field, the primary step before any treatment of medication is testing of disease. Testing is
very necessary to find the real cause behind any symptoms or disease. From a list of different testing
methods in biomedical sciences, q-PCR and Flow cytometry/Elisa are two important methods.
Immunological memory is the ability of immune system to identify the disease causing outer agents.
This memory is responsible for further activation of antibodies to counter that outer attack.
In recent years, we have heard a lot about RT-PCR test. RT- PCR is an abbreviation of reverse
transcription polymerase chain reaction. During COVID-19, almost every person heard about this name.
This test is different from q-PCR, however it is also a kind of PCR.
Flow cytometry is another test that is mostly used to evaluate fluids and their characteristics in our
body. It is necessary to find the details of different constituents in a fluid. Apart from testing, analysis of
the test results is also necessary as at this step we recognize the issues and the important parameters
that are responsible for the cause in our body.
In this study, Flow cytometry, PCR, and ELISA were carried out following the stimulation of cells. All
circumstances had the same statistical significance with P-values range from 0.038589 (0.003) to
0.000034 (0.0193) for each of the studies. CD3/28 and CD3/28/200 naïve cells, on the other hand, show
a substantial increase in TNF-, while CD3 is down-regulated. TNF expression in memory cells was found
to be at the same level in CD3/28 and CD3/28/200 cells. Findings from this study (p value=0.579026,
0.390750, 0.01195, 0.00007) show that there is statistical significance in the presence of the IL2
inflammatory mediators. Naive T cells spanning CD3/28 and CD3/28/200 showed an enhanced
expression of IL-2.
Introduction
Flow cytometry test is used to study the fluid particles of our body Chow (2021). Elisa test is a test to
detect antibodies in human body. Elisa is an abbreviation for enzyme linked immunosorbent assay. Elisa
test is used in diagnosing many diseases like HIV, rotavirus, Zika virus, Syphills etc. Woodall, R. T.
(2020). Flow cytometry is used in research for a number of purposes like cell counting, sorting,
detecting micro-organisms, diagnosing bone marrow and blood cancers etc. These tests are very useful
in detecting and diagnosing problems in human body. All the problems where fluid flow study is
required, flow cytometry is used to determine the parameters and its effects. After complete blood
count (CBC) test, this test is commonly used as a follow-up test Anderson Gwenin, & Gwenin,(2019).
q-PCR test is a quantitative test. q-PCR is an abbreviation for quantitative polymerase chain reaction.
This technology is used for measuring DNA using PCR. PCR is a common method for amplifying DNA.
This test is a modern methodology for studying gene expression. In this test, nucleic acids are detected
rapidly that are responsible for the disease in the body.
Memory T and B cells constitute basis of immunological memory, yet a lot is still unknown about their
biology. This study focuses on T cell responses using a model of antigen stimulation based on
antibody attached to artificial devices. This was a cross sectional study and

conducted at University of Hull. Ethical approval was obtained from Ethical review board. In this study,
memory cells differentiated in vitro from Balb/c mice (spleen and lymph nodes), compared to naïve
cells, freshly harvested from Balb/c mouse spleens and lymph nodes. After stimulation of cells, flow
cytometry, PCR and ELISA were done.
One of CD-200's more common names is MRC OX-2. An average human CD-200 is 41-47 kDa in size. CD-
200 is an intracellular signaling protein with two immunoglobulin superfamily domains. The genes that
code for CD-200 are located on chromosome 3. (3q12-13) Khanal & et.al., (2020). Extracellular
immunoglobulin superfamily domain (single V + C), short cytoplasmic tail, and a single transmembrane
region devoid of signaling motif make up the molecule's structural components. At its N-terminal
immunoglobulin like domain names, CD200's N-terminal GFCC faces are the primary point of contact′
between CD200 and its receptor. Residues E44, I71, T73, E75, and I133, which are typically found in
CD200R1's GFCC′ region, play an important role in this interaction, despite the fact that E44 is positioned
outside of this area, within the so-called A strand, in the V-like domain Zarei & et.al., (2021).
C/EBP- controls CD-200 expression at the transcriptional level. TNF-, IFN-IL-6, and IL-1 are all under their
control, as are a slew of other molecules. CD-200 expression is induced by TNF- and IFN- through NF-
kappa B, STAT1, and IRF-1. When it comes to DNA, human and mouse CD-200 cells share an estimated
81.7% similarity, while the protein content is estimated at 77.5 percent Liu & et.al.,(2018)
Graph pad prism software is a sophisticated software as it contains various tools to interpret and
analyze the data. It is easier to plot curve and data in this software. For analysis, it contains various
inbuilt tools like anova test, parametric test etc. that provides an easy way to do the analysis of data set
(Winter, 2013). In our scenario, we have collected data from two teams for these tests and analyzed
these data set using the graph pad prism software. In addition to analysis, data representation is also
quite easier in graph pad prism software Zhou, T. & et.al., (2022).
Materials and methods
Chemical, Reagents, and buffers
Different materials are required to do these tests. Apart from materials, instruments are needed for
carrying out these test like computer and related apparatus. We will discuss in details about methods
used to do these tests one by one.
Memory cells were differentiated in vitro two/three weeks ahead, by seeding solenocytes with anti-CD3
and anti-CD28 activating antibodies (to simulate signal 1 and 2, respectively) for a week, maintained at
37°C in 5% CO2 in 24-well plates with complete RPMI (penicillin, streptomycin, glutamine and 5% FCS),
then washed and seeded in culture with memory cell cytokines, IL-7 and IL-2 in complete RPMI for
additional 2-3 weeks. Both naïve and memory T cells were used in this as primary cell lines. Microscopy
of different markers i.e., CD28, CD 03, CD3 + CD28, and CD3+CD28+CD200 were also done by using
Neuberger chamber.
Animals
The animals were maintained and used under strict ethical conditions according to international
recommendations for animal welfare set by Committee Members, (International Society on Toxicology,
1992). In this study, memory cells differentiated in vitro from Balb/c mice (spleen and lymph nodes),
compared to naïve cells, freshly harvested from Balb/c mouse spleens and lymph nodes.