User Account

All Pages

Flow cytometry, Biology

Assignment Help:
Ask que1. Design a 4 colour Flow cytometry experiment with an aim to identify B220-, CD3+, CD4+
(DOUBLE POSITIVE) AND B220+, CD3-, CD4- (DOUBLE NEGATIVE) cells. Select from
the following antibody-fluoro chrome choices.
Anti-CD3 FITC (Excitation 488nm, Emission 530nm)
Anti-CD4 PE (Excitation 488nm, Emission 585nm)
Anti-CD4 FITC (Excitation 488nm, Emission 530nm)
Anti-B220 PE CY7 (Excitation 488nm, Emission 780nm)
Anti-B220 PerCP (Excitation 488nm, Emission 690nm)
Anti-B220 PE (Excitation 488nm, Emission 585nm)
i). Provide a rationale for selecting the specific antibody-fluorochromes for your
experiment.
ii). Explain why your experiment will be unsuccessful if you used the wrong
combination of antibody-fluorochromes.
iii). Name the antibody-fluorochrome conjugates that cannot be used together if you are
to achieve the aims of your experiment.
2. Define a gating strategy using the reagent mixture from 1 (above) to determine the
i). percentage of lymphocytes in a total spleen sample
ii). percentage of CD3+ T cells in the lymphocyte population
iii). percentage of CD3+CD4+ T cells in the lymphocyte population
iv). percentage of B cells in the lymphocyte population
3. Shown in the Figure for Question 3 are gating strategies to determine percentages of
Germinal centre B cells. Please note that 7AAD is a chemical substance that binds to
dead cells.
i). List the differences in the 2 strategies that might be the reason for different
proportions of cells.
ii). Indicate the correct strategy to identify these cells (Top panel or Bottom panel).
iii). What would happen if you used the incorrect strategy to gate on the Germinal
centre B cells?
4. Shown in the Figure for Question 4 are the results of an experiment done to determine
percentages of subsets of CD3+CD4+ cells. The subsets are T follicular regulatory (TFR),
T follicular helper (TFH), regulatory T (Treg or REGS) and effector (EFF) T cells.
Based on the results shown the Figure for Question 4 and from your BIOL3144/6144
lectures,
i). List the percentages of these cells as a proportion of total cells processed and
describe the function(s) of TFR, TFH and Treg
ii). Name a key transcription factor (not shown in Figure) that is important for the
development of TFR and Treg cells, and
iii). Name a chemokine receptor and a regulatory molecule that are expressed at high
levels by both TFR and TFH.
5. Using the diagram shown in the Figure for Question 5, describe the population hierarchy
or strategy to identify
i). CD19+ cells
ii). CD4+ T cells
iii). CD8+ T cells, and
iv). eosinophils (Eos)
6. Identify and analyse lymphocyte subpopulations in the lysed peripheral blood and
answer the following Questions after careful analysis of the plots shown in Figure for
Question 6.
i) What is the percentage of lymphocytes in the total cell preparation?
ii) What is the ratio of T helper cells to T cytotoxic/suppressor cells in total T cells?
iii) What is the antibody conjugate used to identify NK cells?
iv) CD19 APC conjugate has been used to determine which population?
v) What could be the other cells, besides lymphocytes in the FSC/SSC plot?
7. The generation of distinct T helper (Th) subsets is driven by the environment (e.g. the
presence of specific cytokines and activation of the JAK-STAT and/or Smad pathways),
and defined by their expression of specific transcription factors and cytokines. These
subsets mediate distinct functions. Flow cytometry can be used to define (phenotype) Th
subsets. Transcription factors, cytokines and cell surface molecules (also referred to as
markers) expressed by these cells can be detected specific fluorochrome-conjugated
antibodies. Based on all your lectures on T lymphocytes and flow cytometry seminars,
complete the information in Table 1. You can either copy Table 1 or generate your own
but complete the information.
8. A flow cytometric technique in which an intracellular fluorescent label is divided equally
between daughter cells upon cell division is applicable to in vitro cell division, as well as
in vivo division of adoptively transferred cells, and can resolve multiple successive
generations. The label is fluorescein derived, allowing monoclonal antibodies conjugated
to other compatible fluorochromes to be used to immunophenotype the dividing cells.
Based on your lectures on T lymphocytes, flow cytometry seminars and also drawing
upon your answers to Question 7, you are to set up an experiment in which you will
measure the proliferation of one Th subset of your choice using mouse splenocytes. The
subset could be Th1, Th2, Th17 or Treg cells.
Describe the experimental set up and how you will measure the various parameters by
flow cytometry using the following points as a guide.
a) Starting cell population – (i) what T cell subset would you use, (ii) how would you
purify them by flow cytometry, (iii) what antibodies would you use and (iv) how would
you ensure they are a pure population?
b) Cytokines used to differentiate the cells – what cytokines would you use to
differentiate the Th subset you want to generate?
c) Detection of cell surface activation markers – how would you achieve this by flow
cytometry and name 3 markers you would use?
d) Detection of transcription factor(s) – name the transcription factor(s) and how would
you detect using flow cytometry?
e) Detection of soluble factors (e.g. cytokines) produced – name the cytokines astion #Minimum 100 words accepted#

Related Discussions:- Flow cytometry

Define reaction for nelson-somogyi method, Define Reaction for Nelson-Somog...

Define Reaction for Nelson-Somogyi Method? The reagent used for enolization contains alkaline solution of cupric sulfate and Rochelle salt. Rochelle salt in the reagent prevent

Determine the muscular-skeletal system, Determine the Muscular-Skeletal Sys...

Determine the Muscular-Skeletal System It is known we all do movement and these take place by movement of muscles in coordination with the skeletal system. In human body muscle

Explain the various types of protein structure, Explain the various types o...

Explain the various types of Protein Structure? Protein Structure :  The structure of proteins can be examined at four levels of increasing complexity, with the primary struc

Explain heat preservation - method of food preservation, Explain Heat Prese...

Explain Heat Preservation - Method of Food Preservation? The process of heating was used centuries before its action was understood. Food is heated up or cooked. Heat kills mi

Nutrition in animals, filter feeding mode of nutrition is observed in- a) a...

filter feeding mode of nutrition is observed in- a) amoeba b) euglena c)paramoecium d) all of the above

Dietary fibre requirement during diabetes, Q. Dietary Fibre requirement dur...

Q. Dietary Fibre requirement during diabetes? Dietary fibre is that part of food which is not digested by the intestines. High fibre meals have shown to give the best glycemic

Explain procedure for acid fast staining, Explain Procedure for Acid Fast S...

Explain Procedure for Acid Fast Staining? Carry out the exercise using the steps enumerated herewith. 1. Label the clean, non-greasy slides and make smears of M. smegmatis,

Bacterial diseases-actinomyces, Actinomyces Members of the genus Actin...

Actinomyces Members of the genus Actinomyces are facultative anerobic, gram-positive, non acid-fast, non-spore forming, nonhaemolytic rods. All Actinomyces require rich media

Growth curve, typical growth curve in plants

typical growth curve in plants

Floristic composition - qualitative characters, Floristic Composition - Qua...

Floristic Composition - Qualitative Characters One of the important qualitative characteristics of a community is its floristic composition. This broadly refers to the kind o

Write Your Message!

Captcha
Order Assignment

Featured Services

Order Now

Popular Subjects

Free Assignment Quote

Assured A++ Grade

Get guaranteed satisfaction & time on delivery in every assignment order you paid with us! We ensure premium quality solution document along with free turntin report!

Submit Assignment

Academics

Major Subjects

Majors

Get In Touch

whatsapp: +91-977-207-8620

Phone: +91-977-207-8620

Email: [email protected]

TERMS & POLICIES

HELP & SUPPORT

All rights reserved! Copyrights ©2019-2020 ExpertsMind IT Educational Pvt Ltd