Medical Delivery Imaging and Drug Analysis: Immune System Report

This report delves into the analysis of inflammatory cytokines using flow cytometry, q-PCR, and ELISA to assess the immune system's response in mice. The study investigates the immunological memory and the role of these testing methods in biomedical research, particularly in understanding T cell responses. Key findings indicate statistically significant changes in TNF- and IL-2 expression in naïve and memory cells under different stimulation conditions (CD3/28 and CD3/28/200). Flow cytometry results highlight differences in cell size and granulation, while ELISA tests reveal higher concentrations of naïve cells compared to memory cells. The analysis was performed using GraphPad Prism software, emphasizing its utility in interpreting and representing complex data sets. The report concludes that these methods are crucial for diagnosing and understanding various diseases and immune-related conditions.

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
n biomedical fi eld the primary step before any treatment of medication is testing of disease esting isI , . T very
necessary to fi nd the real cause behind any symptoms or disease rom a list of different testing. F methods in
biomedical sciences q CR, -P and lowF cytometry lisa/E are two important methods.
mmunologicalI memory is the ability of immune system to identify the disease causing outer agents. hisT
memory is responsible for further activation of antibodies to counter that outer attack.
n recent years we have heard a lot about R CR test R CR is an abbreviation of reverseI , T-P . T- P transcription
polymerase chain reaction. During C V DO I -19, almost every person heard about this name. hisT test is
different from q CR however-P , it is also a kind of CRP .
low cytometry is another test that is mostly used to evaluate fl uids and their characteristics in ourF body.
t isI necessary to fi nd the details of different constituents in a fl uid. 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.
n this study low cytometry CR and SA were carried out following the stimulation of cells AllI , F , P , ELI .
circumstances had the same statistical significance with values range from toP- 0.038589 (0.003) 0.000034
for(0.0193) each of the studies. CD3/28 and CD3/28/200 na veï cells, on the other hand, show a
substantial increase in while CD is down regulated e pression in memory cells was foundTNF-, 3 - . TNF x to
be at the same level in CD and CD cells indings from this study p value3/28 3/28/200 . F ( =0.579026,
show that there is statistical significance in the presence of the0.390750, 0.01195, 0.00007) IL2
inflammatory mediators aive cells spanning CD and CD showed an enhanced. N T 3/28 3/28/200
e pressionx of IL-2.
Introduction
low cytometry test is used to study the fl uid particles of our bodyF Chow (2021) lisa test is a. E test to
detect antibodies in human body lisa is an abbreviation for enzyme linked immunosorbent. E assay lisa. E
test is used in diagnosing many diseases like V rotavirus ika virus Syphills etcHI , , Z , . Woodall, R. T.
(2020) low cytometry is used in research for a number of purposes like cell. F counting sorting detecting, ,
micro organisms diagnosing bone marrow and blood cancers etc hese- , . T tests are very useful in detecting
and diagnosing problems in human body. All the problems where fl uid fl ow study is required fl ow,
cytometry is used to determine the parameters and its effects After. complete blood count C C( B ) test, this
test is commonly used as a follow up- test Anderson Gwenin, & Gwenin,(2019).
q CR test is a quantitative test q CR is an abbreviation for quantitative polymerase chain reaction-P . -P . hisT
technology is used for measuring D A using CR CR is a common method forN P . P amplifying D A hisN . T
test is a modern methodology for studying gene e pression nx . I this test, nucleic acids are detected
rapidly that are responsible for the disease in the body.
Memory and cells constitute basis of immunological memory yet a lot is still unknown about theirT B ,
biology his study focuses on cell responses using a model of antigen. T T stimulation based on antibody
attached to artificial devices. hisT was a cross sectional study and

conducted at niversityU of ullH . thicalE approval was obtained from thicalE review board. nI this study,
memory cells differentiated in vitro from alb c mice spleen and lymph nodes compared to na veB / ( ), ï cells,
freshly harvested from alb c mouse spleens and lymph nodes After stimulation of cells fl owB / . , cytometry,
CRP and SAELI were done.
ne of CD s more common names is MRC An average human CD is kDa in size CDO -200' OX-2. -200 41-47 . -
is an intracellular signaling protein with two immunoglobulin superfamily domains he genes that200 . T
code for CD are located on chromosome q-200 3. (3 12-13) Khanal & et.al., (2020 tracellular). Ex
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 terminal' . N-
immunoglobulin like domain names CD s terminal CC faces are the primary point of contact, 200' N- GF ′
between CD and its receptor Residues and which are typically found in200 . E44, I71, T73, E75, I133,
CD R s200 1' CCGF ′ 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 controls CD e pression at the transcriptional level and are all under their/EBP- -200 x . TNF-, IFN-IL-6, IL-1
control as are a slew of other molecules CD e pression is induced by and through, . -200 x TNF- IFN- NF-
kappa S A and R hen it comes to D A human and mouse CD cells share an estimatedB, T T1, I F-1. W N , -200
similarity while the protein content is estimated at percent81.7% , 77.5 Liu & et.al.,(2018)
raph pad prism software is a sophisticated software as it contains various tools to interpret andG analyze
the data t is easier to plot curve and data in this software or analysis it contains various. I . F , inbuilt tools
like anova test, parametric test etc. that provides an easy way to do the analysis of data set inter(W ,
n our scenario we have collected data from two teams for these tests and analyzed2013). I , these data
set using the graph pad prism software n addition to analysis data representation is also. I , 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 e will discuss in details about methods. W
used to do these tests one by one.
Memory cells were differentiated in vitro two three weeks ahead by seeding solenocytes with anti CD/ , - 3 and
anti CD activating antibodies to simulate signal and respectively for a week maintained at- 28 ( 1 2, ) , C in37° 5%
CO2 in well plates with complete R M penicillin streptomycin glutamine and CS24- P I ( , , 5% F ), then
washed and seeded in culture with memory cell cytokines and in complete R M for, IL-7 IL-2 P I additional
weeks oth na ve and memory cells were used in this as primary cell lines2-3 . B ï T . Microscopy of different
markers i e CD CD CD CD and CD CD CD were also done by using. ., 28, 03, 3 + 28, 3+ 28+ 200 eubergerN
chamber.
Animals
he animals were maintained and used under strict ethical conditions according to internationalT
recommendations for animal welfare set by Committee Members, nternational(I Society on o icologyT x , 1992).
n this study memory cells differentiated in vitro from alb c mice spleen and lymph nodesI , B / ( ), compared to
na veï cells, freshly harvested from alb c mouseB / spleens and lymph nodes.
Mice were bled at and hours and sera were separated and stored at C until0, 1/4, 1/2, 1, 2, 4, 8 24 , −20°

use.
Test Details
q-PCR ( uantitativeQ polymerase chain reaction)
Materials needed: luorescentF dyes, D AN binding dyes, real ti me CRP instrument, CRP analysis software, CRP
prime assays CR plastics and, P , CR reagentsP
Method CR chemicals and unique or custom primers are used to start up amplification reactions An: P .
instrument s proprietary software analyses the acquired data from real ti me CR devices he' - P . T
incorporation of a fl uorescent reporter molecule in each reaction well enables real ti me detection of- CRP
products which increases in fl uorescence as the amount of product D A increases D A binding, N . N - dyes
and fl uorescently labelled primers or probes specific to certain sequences are among the fl uorescence
chemistries used for this purpose Haribabu & et.al., (2019) hermal cyclers fi tt ed with fl uorescence. T
detector modules are used to monitor the fl uorescence signal as it amplifies. nI order to quantify the
quantity of amplicon created in each cycle the fl uorescence detected is correlated to the overall amount,
of amplicon.
Flow Cytometry
Materials needed: luorescentF dye, laser beam, detector consisting of forward scatter and side scatter, fl ow
cytometer blood bone, / marrow sample
A fl ow cell is part of the fl uidics system where the sample fl uid is introduced articles are aligned in a, . P single
stream as they move through a ti ny channel into the laser intercept light beam by using sheath( ) fl uid.
aser interrogation is made possible by hydrodynamic focusing which isolates and analyses aL , single cell.
asersL are the most common light source for the optical system since they produce a precise wavelength
and frequency of light Sharifi & et.al., (2019) his is when at least one laser beam is used to pass. T
through the particles asers come in a wide variety of wavelengths from ultraviolet to far red and in a. L , - ,
wide range of power levels. Any fl uorescent probes that have been attached to antibodies and are
compatible with the laser beam are e cited and produce light or fl uoresce at the designatedx ( )
wavelengths as a result of the laser beam interrogation.
Results

he above bar graph represents the details regarding cell condition Along with this in this CD representT . , 3
the data related to aive and CD and CD single cell was used which was easily separated fromN 3/28 03
each other and appeared as one.
he above bar graph represent the details regarding C Cp e pression of cytokines and genes asT T/ x

compared with C C he stastisctical analysos was done for parametric using sharipo wilks test and oneT/ P . T
wat A VA followed by multiple comporsions was obtained with significance l is observed to beNO , I -2
releated to R to be upregulated and e pressed more above the baseline in CD andHP T1 x 0 3/28
CD condition and activated in both naive and memory cells condition3/28/200 .
C CΔΔ - T/ P e pression of cytokines and genes is compared with thex C CΔ T/ P he statistical analysis was done for. T
parametric using Shapiro wilks test and one way A VA followed by multiple comparisons was obtained with- - NO
significance is observed to be abundantly relative to R housekeeping gene to be upregulated and e pressed, IL-2 HP T1 ( ) x
more above the baseline in CD and CD conditions and activated in both na ve and memory cells(0) 3/28 3/28/200 ï
condition ns not significant. P=***<0.0001; - .
low cytometry graphsF :

t can be said that CD CD were bigger in size than that of CD and CD because whenI 3+ 28+ 3 28
theses two type of cells is combine together it create stimulation and they seems more bigger in the size
but some of the cells are still present as single cell Along with this in case of CD CD CD no single. , 3+ 28+ 200
separate cell was observed in the area as there was clumping of cells bigger and centrally granulated than
those observed above cell line for CD and CD because of these three groups of cells are highly3 28
stimulated.
lisa graphsE :

n conte t ofI x low cytometry test it can be said that it is test that helps in studying the fl uid particlesF
of the body urthermore this test is used in various researches for number of purposes like detecting the. F ,
micro organisms diagnosing problems in human body detecting blood cancers and much more hus all- , , . T ,
the problems where the fl uid fl ow study is needed this test help in determining its parameters and its,
relevant effects Also after the complete count than this test is used for the follow up test n the other hand. , . O
the lisa test is one that provides the report through detecting the antibodies that are present within theE
body and is very efficient in detecting various diseases such as V ika virus rotavirus and much moreHI , Z ,
hus from the above graph it can be said that concenetration of the na ve cells is higher than the memoryT , ï
cells which have only the lsight increase Moreover higher increase in the naiev cells is good as it is. ,
considered as essentail components that helps the body to fi ght off new and unrecognized infections in the
body ence through higher cobecentartion of the such na ve cellas the body would be able to recognize. H , ï
and repsosnd to the new pathogens