1. Potency of agonists and antagonists
Medsci 303 Lab 2
Potency of agonists and antagonists
Take a look at your station – you will need to leave it as you find it, so familiarize yourself with the location of the timer, the pipettes, etc.

2. How is the tissue suspended?
Tissue Hook 2 to Transducer
Carbogen bubbles
(ensure these going now)
Tissue/Carbogen Hook 1
Tissue
Resting tension across
tissue is 0.5gm
TIMER STARTED?

3. Tips for the lab
Please do not touch the equipment on your bench during this introduction (other than washing every 5 min)
Do not close the Chart program, log out, or do anything which will undo the calibration that has been done for your station
You will be writing up this lab – use your time in the lab well (Report Guide is on website and data will be posted on Cecil)

4. Plan for today’s lab Introduce demonstrators
Brief intro to this lab and organ baths
Prepare dilutions
Conduct the experiment
Measure/transform the responses
Record your data. Submit to demonstrator.
Clean up. Follow instructions or lose marks.

5. Today’s Health And Safety
Wear gloves and safety glasses
There are 3 types of rubbish bins:
Small YELLOW buckets on your bench (tips, empty tubes, gloves) for during experiment
Big YELLOW bins for lab waste too
Paper waste bins under the bench (for paper trash NOT for anything used in the lab, especially not gloves)

6. Intro to the Power Lab Equipment
We have already:
Set up Chart for Windows
Opened GP Ileum Method
Auto Calibrated the Powerlab to 0.5g
(calibration “translates” the contraction into a gram value)
DO NOT CLOSE PROGRAM or it will need to be done again

7. Intro to Organ Baths
Designed for studying isolated tissue - a pharmacology screening tool to determine the concentration-response relationship in a contractile tissue
Keep tissue alive by providing
Nutrients in the Krebs solution
Carbogen gas (95% O2 / 5% CO2) (keep an eye on these bubbles during the lab)
Warmth via heated Krebs solution

8. Intro to the Organ Bath – Surrounding Apparatus
B) Do Not Touch!
C) Unhook from force transducer to suspend tissue
A) Adjust resting tension
D) Loosen to remove carbogen hook
Tissue hooks

9. Equilibrate tissue 30 mins
Today's experiment
Using guinea pig ileum in an organ bath to gain data to construct concentration-response curves
Comparing and contrasting the curves of 2 agonists (Exp A)
Observe the effect on the concentration-response curve in the presence of 2 different antagonists (Exp B/C)
Equilibrate tissue
30 mins
Add agonist
(5 min cycle x7)
Add antagonist (B/C)
Equilibrate tissue 30 mins
0 mins
30mins
65 mins
95 mins
135 mins
Suspend tissue
Gather data and post

10. Ask yourself? WHICH EXPERIMENT AM I DOING? A? B? or C?
Read the protocol through completely while equilibrating your tissue
Ask yourself?

11. Agonist Dilutions
Use the correct pipette / tip combination – ask if you are unsure
We suggest:
Prepare 500 ul of each agonist solution you need. i.e. (V2 = 0.5 ml)
You need to:
Use water as your diluent.
If your drug is on ice, keep your dilutions on ice.

12. Before you start adding drugs...
Check tension (big silver dial), have you equilibrated for 30 minutes (washing every 5 min)?
Do you know how to use Chart?
Enter time/drug conc. => add drug => hit enter on chart to mark time)?
Prepare your agonist serial dilutions:
4 test tubes per agonist
Add conc. from low to high (i.e first)
Keep pipette tips below the Krebs when adding agonist

13. 5 minute cycle Add drug Add next conc. Empty and refill @ 0min @ 30secW
Readjust tension
~ 4mins

14. Start adding your first drug…
If you are unsure of what you are doing...think first...ask second...
Start adding your first drug…

15. 5 minute cycle Add drug Add next conc. Empty and refill @ 0min @ 30sec
Readjust tension W W @
~ 4mins

16. For part two:
Exp A
When last contraction is ending, drain and refill chamber twice, start timer for 30 minute equilibration, washing every 5 minutes
Exp B & C
Drain and refill the reservoir – Set the tap to overflow to drain
When the reservoir is empty, STOP overflow (or you will waste the new Krebs)
Add Krebs with antagonist
Drain and refill the chamber twice, start timer for 30 minute equilibration, washing every 5 minutes

17. Parts of a Laboratory Report
MEDSCI 305
Pharmacology Undergraduate Student

18. Support for Reports Report Writing Guide Rubric Website Piazza Tutors
Group

19. Lab Reports Aim Introduction Method Results Discussion Conclusion
Referencing (primarily in Intro & Discussion)
PRESENTATION

20. Introduction Scientist vs Undergraduate Student
“To learn something about the science of the course you are taking”
An effective introduction in a lab report
Established the learning context for the lab
Link practical with lectures (Lecture 3, Chapter 4 Rang and Dale)
General background information
Your scientific writing skills are important here. A place to assess conciseness, flow of information as well as relevance of information

21. Materials and Methods What did you do and how did you do it?
Animal species, tissue type/origin
Experimental setup/process description
Equipment used (organ bath, analysis or graphical software)
Drugs and Solutions (final conc of exposure not how each working solution is made)
Data Analysis
Which data are from which experiment
Data manipulation, how was it transformed?
Include an appendix to highlight which data excluded

22. Results Display your graphs using the format provided on the web site.
Class data
Narrative results also required, provide quantitative values where possible so magnitude of effect can be clearly seen
Think about flow/layout here too

23. Discussion 3 pages maximum
Answer the questions given to you specifically
Depth of answer important
Results based support where possible
Excellent referencing of facts

24. Measuring contractions
Stop the trace
Move the M pointer to the baseline before the first contraction you want to measure.
Move your mouse along the line and record the maximum response (g) within the ~30 second window.
Move M to the baseline just prior to the next agonist addition and measure the maximum. Do this for each concentration.
Change all your contractions into % of maximum, i.e. if your biggest contraction is 8g, then make all other contractions a % of 8g. 4g = 50% etc…
Record your data on the handouts/in your manual

25. Normalising Responses
Change all your contractions into % of maximum
𝐶𝑜𝑛𝑡𝑟𝑎𝑐𝑡𝑖𝑙𝑒 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 x 100%
e.g. if your biggest contraction is 8g, then a 4g response =
4 8 𝑥 100%=50%

26. Class Data Record your data into the data sheet in your manual NOW
MAKE SURE IT IS CLEAR SO YOUR DEMONSTRATOR CAN UNDERSTAND IT.
Class data will be posted on Cecil by 5pm Thurs for you to process and present in your report. You can get started on the rest of the report earlier – there is plenty to do!

27. Continue with part two…
Equilibrate tissue
30 mins
Add agonist
(5 min cycle x7)
Add antagonist (B/C)
Equilibrate tissue 30 mins
0 mins
30mins
65 mins
95 mins
135 mins
Suspend tissue
Gather data and post
Add drug
Add next conc.
Empty and refill @
0min
@ 30sec
1min 30sec
5mins
Readjust tension
~ 4mins

28. Clean-up instructions
Empty petri dishes and rinse them under the tap
Test tubes emptied in sink & disposed of in waste bin
Tissue disposed of in waste bin
Upper reservoir drained, then filled with 2 litres of hot water (leave on overflow), leave bubbles flowing
Tidy up your bench (pipettes etc.) – leave it as it was !
Empty your ice bucket into sink
Do not save chart file when closing Chart, log out
Tidy away your mouse and keyboard etc.
Station must be checked off by a tutor/demonstrator before you leave.