Medsci - PowerPoint Presentation

Slide1

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.Slide2

How is the tissue suspended?

Tissue

/Carbogen

Hook 1

Tissue

Hook 2

to Transducer

Carbogen bubbles

(ensure these

going now)

Tissue

Resting tension across

tissue is 0.5gm

TIMER STARTED?Slide3

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)Slide4

Plan for today’s lab

Introduce demonstrators

Brief intro to this lab and organ baths

Prepare dilutionsConduct the experiment

Measure/transform the responsesRecord your data. Submit to demonstrator.

Clean up. Follow instructions or lose marks.Slide5

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 experimentBig 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)Slide6

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 Slide7

Intro to Organ Baths

Designed for studying isolated tissue

- a pharmacology screening tool to determine the concentration-response relationship in a contractile tissueKeep 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 solutionSlide8

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 transducer

to suspend tissue

Tissue hooksSlide9

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 mins95 mins135 minsSuspend tissueGather data and postSlide10

Ask yourself?

WHICH EXPERIMENT AM I DOING?

A? B? or C?Read the protocol through completely while equilibrating your tissueSlide11

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.Slide12

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 agonistAdd conc. from low to high (i.e. 10

-6 first)Keep pipette tips below the Krebs when adding agonistSlide13

5 minute cycle

Add drug

Add

next conc.

Empty and refill

@

0min

@ 30sec

@

1min 30sec

@

5

mins

W

WEmpty and refill

Readjust tension

@~ 4minsSlide14

Start adding your first drug…

If you are unsure of what you are doing...think first...ask second...Slide15

5 minute cycle

Add drug

Add

next conc.

Empty and refill

@

0min

@ 30sec

@

1min 30sec

@

5

mins

W

WEmpty and refill

Readjust tension

@~ 4minsSlide16

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 drainWhen the reservoir is empty, STOP overflow (or you will waste the new Krebs)

Add Krebs with antagonistDrain and refill the chamber twice, start timer for 30 minute equilibration, washing every 5 minutesSlide17

Parts of a Laboratory ReportMEDSCI 305

Pharmacology Undergraduate StudentSlide18

Support for Reports

Report Writing Guide

Rubric

WebsitePiazzaTutors

GroupSlide19

Lab Reports

Aim

Introduction

MethodResultsDiscussion

ConclusionReferencing (primarily in Intro & Discussion)

PRESENTATIONSlide20

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 labLink practical with lectures (Lecture 3, Chapter 4 Rang and Dale)

General background informationYour scientific writing skills are important here. A place to assess conciseness, flow of information as well as relevance of informationSlide21

Materials and MethodsWhat 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 experimentData manipulation, how was it transformed?Include an appendix to highlight which data excludedSlide22

ResultsDisplay 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 tooSlide23

Discussion

3 pages maximum

Answer the questions given to you specifically

Depth of answer importantResults based support where possibleExcellent referencing of factsSlide24

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 manualSlide25

Normalising Responses

Change

all your contractions into % of

maximum

x 100%

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

 Slide26

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! Slide27

Continue with part two…

Equilibrate tissue

30 mins

Add agonist

(5 min cycle x7)

Add antagonist (B/C)

Equilibrate tissue 3

0 mins

Add agonist

(5 min cycle x7)

0 mins

30mins

65 mins

95 mins

135 minsSuspend tissueGather data and postAdd drug

Add next conc.

Empty and refill@0min@ 30sec@ 1min 30sec@5mins

Empty and refill

Readjust tension

@

~

4

minsSlide28

Clean-up instructions

Empty

petri

dishes and rinse them under the tapTest 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 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.