Follow up the document and audio recording it explains exactly what to
do. Follow it up what it says and create graph for it as well. Havard
reference should be used.https://drive.google.com/file/d/1jvER-G-bn4Tr9s1Qg…6PHYM004W: Practical
Safety: This method involves the use of Griess reagents 1 and 2 (containing
sulphanilamide and napthylethyl-endiamide dihydrochloride and H3PO4
acid respectively). Use appropriate protective clothing i.e. lab coats, eye
protection and gloves. Follow the general safety instructions given to
you in the laboratory.
Background: Nitrite will be assayed by using a spectrophotometric method.
Griess reagent, containing sulphanilamide and napthylethyl-endiamide
dihydrochloride, gives a purple/deep red colour when added to a sample
containing nitrite. The intensity of the colour is measured at 550 nm on a plate
reader to determine the concentration of nitrite in each of the samples. You
are provided with samples generated from an in vitro cell culture screen of 5
novel therapeutics (samples 1-5) for the treatment of a broad range of
inflammatory pathologies. You are also provided with a placebo and a sample
utilising the glucocorticoid prednisone.
Prior to attending session: You should review the mechanism of action
of prednisone, role of nitrite, neutrophil cell migration and the
CXCL1murine chemokine KC in inflammation. A ref is provided to cover
aspects of this http://www.jimmunol.org/content/180/6/4308
Reagents: You are provided with Griess 1 reagent (1 g of sulphanilamide
added to 95 ml distilled water and 5 ml H3PO4) and Griess 2 reagent (0.5 g of
napthylethyl-endiamide dihydrochloride added to 100 ml distilled water). This
reagent was made up 24 h prior to use and stored in light protective bottles at
Part A – Screening test for nitrite
Before determining the concentration of nitrite, you need to first ascertain
which samples have nitrite present in them. The initial stage in the
investigation is therefore to carry out a qualitative test on the samples.
1. Prior to determination of nitrite in the samples, aliquot equal volumes of
Griess 1 to Griess 2 and leave for ten minutes to equilibrate.
2. To seven separate plastic tubes add samples as below:
100 µl of sample 1
100 µl of sample 2
100 µl of sample 3
100 µl of sample 4
100 µl of sample 5
100 µl of sample 6 (placebo)
(vii) 100 µl of sample 7 (prednisone)
3. To each add: 100 µl of mixed Griess 1 and Griess 2 solution.
4. Create your own scale for determination of colour intensity and note
the intensity/colour change observed. Indicate whether nitrite is present
or absent from the sample.
Qualitative Results provided:
Samples 1-7 in chronological order starting with arrow
Part B – Nitrite standard curve
1. Weigh out Sodium Nitrite (RMM 69) and make a stock solution. Take
an aliquot and dilute to a working concentration of 400 µM. (THINK!
Remember you don’t need to make litres of a reagent when you
can dilute something much more concentrated to your desired
concentration, meaning you don’t waste precious reagents).
Result Provided: 13.6 mg has been weighed out
2. Make a nitrite standard curve (8 points) with 400 µM (point 1) as your
top standard and DMEM (vehicle) as your 0 µM (blank, point 8) using
serial 1:1 dilutions. Remember to show workings out in your report.
3. Add 100 µl of each standard to a 96 well plate in duplicate.
4. Add 100 µl of unknown samples to the 96 well plate in triplicate.
5. Add an equal volume of complete Griess reagent (Griess 1 and Griess
2) to sample and standards. (Hint: It’s best to add to the top standard
first it should go dark purple indicating that the reagent is working).
6. Shake/tap plate to ensure mixing and read on a plate reader at 550 nm.
Part B Quantitative Results Provided for Greiss assay (Raw excel
Standard samples: A5/6 is 400 µM and serial 1:1 dilution undertaken,
H5/6 is 0
Part C- in vivo data
1. Following the in vitro screen (Part A and B), the novel therapies
have been tested in vivo using a 4h zymosan peritonitis model. The
following results have been generated from peritoneal lavage fluid
utilising cell counting and ELISA.
Table X. in vivo data.
4.2 ± 0.7
1.5 ± 0.6
4.1 ± 0.6
1.2 ± 0.2
5.4 ± 2.1
6.5 ± 1.1
1.0 ± 0.2
473 ± 81
211 ± 37
521 ± 42
87 ± 7
1193 ± 58
1519 ± 63
100 ± 10
For your write up:
Complete the laboratory report proforma using the sections indicated (i.e. Title,
Aims etc.-do not write the report as one long paragraph) below in Arial 12
and with a maximum of eight pages including Appendix 1 (data print out) and
2 (standard curve/graph).
1. Title: Relevant and informative (2 marks)
2. Aims: Explain why the study is being done and what you are trying to
determine. (2 marks=2 aims)
(1) What is the basis of the Greiss assay commenting on its sensitivity
and alternative approaches (2 marks)
(2) Create your own Intensity coloration scale to enable you to describe
the results observed for the qualitative study (2 marks)
(3) Give a brief description of how you would set up a zymosan
peritonitis model of inflammation and obtain the neutrophils for downstream analysis (4 marks)
4. Results 1 Qualitative: Describe and interpret the results obtained for
each sample from the qualitative assay (3 marks)
5. Results 2 Quantitative
(1) Calculate how you would make up 400 µM stock solution of nitrite
using the amount weighed out for you. Then show how you would
make up the standard curve: remember your units and the dilutions
that you have done (3 marks)
(2) Using the excel data provided in the quantitative results section,
please draw a graph for your standard curve (marks will be allocated
for annotation and correct labelling of axis and plotting of data (4
(3) Calculate from your standard curve the concentration of nitrite
present in the samples display this data in a suitable table. (7 marks
for correctly reading off values/working out mean +/-SD for each
sample, placebo and prednisone).
(4) Rank the drugs in order of effectiveness at inhibiting nitrite release
in the samples-display data in table form. (Most effective =1; Least
effective =7). Indicate which drugs are effective at inhibiting nitrite (Yes
or No). Show results in table form (3 marks).
(5) Describe the results in detail that you have seen in this section (Part
B) and those in Part C (14 marks)
(6) Write a suitable figure legend for the data presented in Part C (4
Explain how nitrite is produced during the inflammatory process. List
other diseases where nitrite is relevant citing sources
Describe the normal physiological role of NO
Explain the purpose of including the placebo and prednisone in the
Suggest possible MofA for the drugs that lower NO
Briefly discuss ideas for improving the experimental design (excluding
mistakes in pipetting etc) and explain what additional information
these changes would provide.
Write a 1-2-line conclusion (20 marks)
7. Refs (5 marks in Harvard style-maximum of fifteen)
y = 0.007x + 0.0212
R² = 0.9987
Purchase answer to see full
Why Choose Us
- 100% non-plagiarized Papers
- 24/7 /365 Service Available
- Affordable Prices
- Any Paper, Urgency, and Subject
- Will complete your papers in 6 hours
- On-time Delivery
- Money-back and Privacy guarantees
- Unlimited Amendments upon request
- Satisfaction guarantee
How it Works
- Click on the “Place Order” tab at the top menu or “Order Now” icon at the bottom and a new page will appear with an order form to be filled.
- Fill in your paper’s requirements in the "PAPER DETAILS" section.
- Fill in your paper’s academic level, deadline, and the required number of pages from the drop-down menus.
- Click “CREATE ACCOUNT & SIGN IN” to enter your registration details and get an account with us for record-keeping and then, click on “PROCEED TO CHECKOUT” at the bottom of the page.
- From there, the payment sections will show, follow the guided payment process and your order will be available for our writing team to work on it.