Medsci 303Lab 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.

How is the tissue suspended?

Tissue/Carbogen Hook 1

Tissue Hook 2to Transducer

Carbogen bubbles(ensure

these going now)

Tissue

Resting tension acrosstissue is 0.5gm

TIMER STARTED?

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)

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.

Today’s Health And Safety

• Wear gloves and safety glasses • There are 3 types of rubbish bins:

1. Small YELLOW buckets on your bench (tips, empty tubes, gloves) for during experiment

2. Big YELLOW bins for lab waste too3. Paper waste bins under the bench

(for paper trash NOT for anything used in the lab, especially not gloves)

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

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 providing1. Nutrients in the Krebs solution2. Carbogen gas (95% O2 / 5% CO2)

(keep an eye on these bubbles during the lab)3. Warmth via heated Krebs solution

Intro to the Organ Bath – Surrounding Apparatus

A) Adjust resting tension

D) Loosen to remove carbogen hook

B) Do Not Touch!

C) Unhook from force transducerto suspend tissue

Tissue hooks

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

Add agonist (5 min cycle x7)

0 mins 30mins 65 mins 95 mins 135 mins

Suspend tissueGather data and post

ASK YOURSELF?

WHICH EXPERIMENT AM I DOING?

A? B? or C?

Read the protocol through completely while equilibrating your tissue

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.

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. 10-6 first)– Keep pipette tips below the Krebs when adding

agonist

5 minute cycle

Add drug

Add next conc.

Empty and refill

@0min

@ 30sec

@ 1min 30sec

@5mins

WW

Empty and refill

Readjust tension

@~ 4mins

START ADDING YOUR FIRST DRUG…If you are unsure of what you are doing...think first...ask second...

5 minute cycle

Add drug

Add next conc.

Empty and refill

@0min

@ 30sec

@ 1min 30sec

@5mins

WW

Empty and refill

Readjust tension

@~ 4mins

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

Parts of a Laboratory Report

MEDSCI 305Pharmacology Undergraduate

Student

Support for Reports

• Report Writing Guide

• Rubric

• Website

• Piazza

• Tutors

• Group

Lab Reports• Aim

• Introduction

• Method

• Results

• Discussion

• Conclusion

• Referencing (primarily in Intro & Discussion)

• PRESENTATION

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

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

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

Discussion

• 3 pages maximum

• Answer the questions given to you specifically

• Depth of answer important

• Results based support where possible

• Excellent referencing of facts

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

Normalising Responses• Change all your contractions into % of

maximum

x 100%

e.g. if your biggest contraction is 8g, then a 4g response =

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!

CONTINUE WITH PART TWO…

Equilibrate tissue 30 mins

Add agonist (5 min cycle x7)

Add antagonist (B/C)Equilibrate tissue 30 mins

Add agonist (5 min cycle x7)

0 mins 30mins 65 mins 95 mins 135 mins

Suspend tissueGather data and post

Add drug

Add next conc.

Empty and refill

@0min

@ 30sec @ 1min 30sec

@5mins

Empty and refill Readjust

tension

@~ 4mins

Clean-up instructionsEmpty petri dishes and rinse them under the tapTest tubes emptied in sink & disposed of in waste binTissue disposed of in waste binUpper reservoir drained, then filled with 2 litres of hot

water (leave on overflow), leave bubbles flowingTidy up your bench (pipettes etc.) – leave it as it was !Empty your ice bucket into sinkDo not save chart file when closing Chart, log outTidy away your mouse and keyboard etc. Station must be checked off by a tutor/demonstrator

before you leave.