Nutrient Sensing by Gprotein Coupled Receptors Assignment | Essay Help Services

Use the sample data to process the lab report
Use the margins and size given in the instructionNUTM3001
Introductory Nutrition
and Metabolism
Lab Manual: Nutrient Sensing by Gprotein
Coupled Receptors
Weeks 10-12, Semester 1, 2020
School of Life and Environmental Sciences
Name:
SID:
If found, please return to:
SOLES Education Support
Room 519, Carslaw Building (F07)
The University of Sydney NSW 2006
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 2
Table of Contents
Introduction ……………………………………………………………………………………………………………….. 3
Aims ………………………………………………………………………………………………………………………… 3
Learning outcomes …………………………………………………………………………………………………….. 3
Associated tutorials ……………………………………………………………………………………………………. 3
Learning schedule ……………………………………………………………………………………………………… 3
Assessment ………………………………………………………………………………………………………………. 3
Safety and logistics ………………………………………………………………………………………………………. 4
What to bring to each laboratory class …………………………………………………………………………. 4
Risks and Hazards ………………………………………………………………………………………………………. 4
Work, Health and Safety guidelines and regulations ………………………………………………………. 4
Background Information ……………………………………………………………………………………………….. 5
The central dogma of cellular signalling ………………………………………………………………………… 5
Calcium sensing receptor (CaSR) ………………………………………………………………………………….. 5
AlphaScreen Technology …………………………………………………………………………………………….. 6
Practical class 1: Cell culture and pipette check ………………………………………………………………… 8
General outline …………………………………………………………………………………………………………. 8
Flow chart ………………………………………………………………………………………………………………… 8
Subculturing (passaging) cells ……………………………………………………………………………………… 9
Pipette check …………………………………………………………………………………………………………… 10
Practical class 2: Effect of calcium on CaSR stimulated phosphorylation of ERK1/2 ……………….. 12
General outline ……………………………………………………………………………………………………….. 12
Flow chart ………………………………………………………………………………………………………………. 12
Cell Preparation (serum starvation) – Laboratory Staff …………………………………………………. 13
Student Procedures ………………………………………………………………………………………………….. 13
Practical class 3: Effect of calcium, phenylalanine and cinacalcet on (1) wild-type CaSR (wtCaSR)
or 2) mutant (S169T) CaSR stimulated ERK1/2 phosphorylation …………………………………………. 16
General outline ……………………………………………………………………………………………………….. 16
Flow chart ………………………………………………………………………………………………………………. 16
Materials and reagents …………………………………………………………………………………………….. 16
Procedure ……………………………………………………………………………………………………………….. 17
Report assessment guidelines ……………………………………………………………………………………… 20
GraphPad Prism Exercise …………………………………………………………………………………………….. 22
Additional questions to think about ……………………………………………………………………………… 24
Appendix – Preparation Room Procedures …………………………………………………………………….. 25
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 3
Introduction
Brought to you by Prof. Arthur Conigrave and Dr James Krycer (with thanks to Dr Vimesh Avlani)
Aims
To test the sensitivity of the calcium-sensing receptor (CaSR) to Ca2+ ions, L-amino acids, and other
calcimimetics. Students will work in pairs to achieve this, by performing experiments with cultured
HEK293 cells that express either the wild-type CaSR or a mutated version of CaSR. Phosphorylation
of Extracellular signal Regulated protein Kinase (ERK1/2) at Thr202-xxx-Tyr204 in response to CaSR
activation will be measured using an ‘Alpha screen’ assay.
Learning outcomes
At the conclusion of this series of practical classes, the student is expected to:
1. Demonstrate familiarity with the principles of nutrient-sensing mechanisms
2. Describe the role of G protein-coupled receptors (GPCRs) in nutrient sensing
3. Describe how GPCRs can respond to distinct nutrient activators with specific signalling responses
4. Describe the nature of biased signalling by different nutrients and its implications for functional
outcomes e.g. digestion, absorption, hormone secretion, etc.
Associated tutorials
Lab de-brief session: Week 12, Tuesday 11-12 – online ALL STUDENTS
Learning schedule
Practical class Activity Description
Practical class 1
Cell culture, pipette testing
Lab 1 • Cell culturing techniques
• Pipetting techniques
Practical class 2
CaSR mediated ERK1/2
phosphorylation
Lab 2 • Introductory tutorial
• Serum starve wt CaSR cells (prior to practical class)
• Investigate effect of Ca2+ concentration on ERK1/2
phosphorylation
Practical class 3
Effects of Ca2+, L-Phe
and the ‘calcimimetic’
cinacalcet
Lab 3 • Discussion, planning experiments
• Investigate effect of calcium, L-phenylalanine, and
cinacalcet concentration on ERK1/2
phosphorylation in (1) HEK-CaSR cells or (2) mutant
HEK-CaSR (S169T or alternative mutant) cells
Preparation for report
writing
Wed
W12
• Data analysis
• Discussion of results
Assessment
This practical will be assessed by a report worth 10% of the total assessment for NUTM3001. Details
at the back of these notes and the Unit of Study Manual.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 4
Safety and logistics
What to bring to each laboratory class
• A copy of this manual (either online or hard-copy)
• A laboratory coat
• A pair of safety glasses
• Enclosed shoes
• A marker pen for writing on plasticware
Risks and Hazards
Work, Health and Safety guidelines and regulations
In addition to the information contained this practical outline, please consult the Unit of Study
manual and Canvas website for additional information.
Risks and hazards in this practical:
In this practical you will handle hazardous substances and perform the following hazardous tasks
/ handle the following pathogens:
• Cell culture (low-risk, biological/chemical)
• Analysing cell extracts using the Alpha Screen assay (low-risk, biological/chemical)
You must observe all required safety precautions. Your unit-coordinator / demonstrator will give
you a safety induction at the beginning of the class that you must attend. If you miss the safety
induction (e.g. you come too late to class) you are not permitted to attend the class.
You can find a detailed Risk Assessment on the Canvas website.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 5
Background Information
The central dogma of cellular signalling
Calcium sensing receptor (CaSR)
The key protein that we will be experimenting with in this practical is the CaSR. As you can guess
from its name, it plays a key role in sensing calcium levels outside of the cell.
Why is it important to sense Ca2+ levels?
Basic facts about the CaSR
• Signal:
o What signals does the CaSR respond to?
• Receptor:
o CaSR
• Effector:
o G protein signalling: phospholipase C (PI → IP3 + DAG), adenylyl cyclase (ATP → cAMP)
o ERK1/2 signalling (what we will be measuring in this practical).
• Response:
o What cellular responses are triggered by the CaSR?
Structure and topology
The CaSR is a huge protein, made up of ~1000 amino acids (residues). Let’s take a look at CaSR in
the UniProt database: http://www.uniprot.org/uniprot/P41180
Scroll down to the Subcellular Location section. Where is the protein found?
Look at the numbers in the Topology section (on Uniprot) and the diagrams on the next page of this
manual.
Why is so much of the protein (~600 residues) found outside of the cell?
The intracellular domain is also quite large (200 residues). Why?
Every cell in our body responds to changes in its
external environment using signalling pathways.
Each pathway has the following components:
Signal → Receptor → Effector → Response
The diagram on the left shows the epinephrine
signalling pathway.
• Identify the signal, receptor and effector.
• What is the response?
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 6
It is now widely appreciated that the CaSR turns on different signalling pathways when it
encounters different ligands. How is CaSR designed to be able to do this?
In this practical, we will be using different ligands and versions of the CaSR (wildtype and
mutant) to explore how the CaSR responds to changes in extracellular Ca2+ levels. How can we
measure CaSR ‘activity’?
AlphaScreen Technology
In this practical, we will be measuring an effector of CaSR, the kinase ERK1/2 (aka ERK1 and ERK2).
CaSR activates this kinase by stimulating phosphorylation at two sites, Thr202-Xxx-Tyr204 (T-X-Y). We
will be measuring this phosphorylation using Amplified Luminescent Proximity Homogeneous Assay
(ALPHA) technology.
AlphaScreen technology can be used to detect numerous protein interactions and
modifications (e.g., phosphorylation) with great sensitivity. The technique uses the concept of
‘antibody-based bead proximity’. This involves two antibodies – to detect ERK1/2 phosphorylation,
one antibody recognises a peptide sequence on ERK1/2 (binding to all ERK1/2 molecules) and a
second antibody recognises the specific phosphorylated site on ERK1/2 (binding only to
phosphorylated ERK1/2 at pT-X-pY).
Each antibody is conjugated to a specific type of bead (donor or acceptor). Exposure of the
donor beads to light of a specific wavelength results in emission of singlet O2, which has a short half-
Top left: Hofer AM, Brown EM (2003) Extracellular calcium
sensing and signalling. Nat Rev Mol Cell Biol, 4(7):530-8.
Top right: Brown EM, MacLeod RJ (2001) Extracellular calcium
sensing and extracellular calcium signalling. Physiol Rev.
81(1):239-297.
Bottom left: Leach K, Conigrave AD, Sexton PM, Christopoulos A
(2015) Towards tissue-specific pharmacology: insights from the
calcium-sensing receptor as a paradigm for GPCR
(patho)physiological bias. Trends Pharmacol Sci, 36(4):215-25
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 7
life and a limited penetration distance (~0.1 μm) in aqueous solution. However, if acceptor beads
are held in proximity to donor beads (they have bound to the same phosphorylated ERK1/2 protein),
singlet O2 released by donor beads excites the release of light at a distinct wavelength from acceptor
beads. The plate reader detects the emitted light so that changes in the phosphorylation state of
the target protein can be quantified.
Here are some questions for you to test your understanding of the AlphaScreen assay:
a. Overall, this assay is a great tool for measuring protein phosphorylation. The old-skool
alternative is ‘Western blotting’. What are the advantages/disadvantages of the AlphaScreen?
b. Oh no! James added the beads with the Tp-X-Yp antibody, but forgot to add the beads with the
ERK antibody. What can James do to rescue this situation?
c. Not again! James forgot to add Al-foil during the incubation. What can James do to rescue this
situation?
d. James wants to see if Ca2+ affects the ability of CaSR to bind to ERK. How could he alter the
alpha-screen assay to measure the binding between these two proteins?
Reproduced from the PerkinElmer
AlphaScreen SureFire Assay kit
manual.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 8
Practical class 1: Cell culture and pipette check
General outline
This class is all about familiarising yourself with the techniques that will be used in this practical,
which will be used in following weeks, will need to be described in the report, and are highly
examinable in the final practical exam.
Students will be working in pairs to:
• Passage cells
• Test pipettors
• Work through the questions in the Background Information section at the start of the manual.
Flow chart
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 9
Subculturing (passaging) cells
This class provides an introduction to cell culture techniques. The key step involves the release of
cells that are adherent to the plastic surface of culture flasks using trypsin-EDTA (0.05%/0.1 M).
Trypsin is a protease that cleaves proteins at the extracellular surface. EDTA (ethylene diamine tetraacetic
acid) is a chelator of divalent cations (primarily Ca2+ and Mg2+ ions), which support proteinbased
interactions with the plastic surface. Exposure of the cell monolayer to trypsin and EDTA leads
to the release of the cells into the medium from where they can be readily recovered by
centrifugation. You will then count these cells and seed 20,000 cells per well into a 96-well plate.
Please note that all handling of cells must be done inside the Laminar Flow (Class II Biosafety)
Cabinets within the X-lab, as indicated in the steps below.
The steps in the procedure are performed in pairs as follows:
I. Biosafety Cabinet: Remove culture medium from your small (T25) flask using an automated
pipette and add 2 mL of medium containing trypsin-EDTA. Transfer the flask to the 37°C CO2
incubator for 5 min. After this time return the flask to the laminar flow cabinet and holding it
horizontally (cell layer at bottom), gently rock it so that the trypsin-EDTA solution slowly
washes back and forth. As the cell layer strips off the plastic you should be able to identify
patches that are clear (no cells) and patches that remain cloudy (cells still attached). If the cells
are not completely detached after 30 s of gentle agitation, return the flask to the incubator for
a further 2 or 3 min. Once the cells are completely detached, and the trypsin-EDTA solution is
cloudy due to the presence of cells, proceed to the next step.
II. Biosafety Cabinet: Add fresh medium containing fetal bovine serum (10 mL) to stop further
proteolysis and transfer the cell suspension to a 50 mL centrifuge tube using the automated
pipette.
III. Centrifugation (1,500 rpm; 3 min): the tubes prepared by several groups will be centrifuged
together in the Elmi Skyspin.
IV. Biosafety Cabinet: Carefully decant supernatants from centrifuge tubes and discard to the
waste container.
V. Biosafety Cabinet: Gently resuspend the cell pellet in 2 mL fresh medium containing fetal
bovine serum. Transfer a 100 μL aliquot to a 1.5ml Eppendorf tube.
VI. Cell counter: Mix 15 μL cells with 15 μL Trypan blue dye on the slide and pipette 10-12 μL of
this mixture into the chamber. Wipe off the excess with a paper towel. Count in the cell
counter, taking note of the concentration of living cells. Repeat this process to get replicate
counts.
VII. Calculations: You need to seed 20,000 cells per well into 32 wells (4 columns of 8 wells each)
in a 96-well plate, in 200 μL per well. How many cells do you need? What volume should they
be resuspended in? Check with your demonstrator.
VIII. Biosafety Cabinet: Dilute out your cell suspension according to your calculations. You can
either dispense the cells into each well one at a time or (if available) pour the cell suspension
into a sterile trough and use the 8-channel pipette to dispense into a column of wells at a time.
IX. Label your plate with your initials and transfer to the CO2 incubator.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 10
Pipette check
Practical classes 2 and 3 require accurate pipetting skills with several different types of pipettes in
96-well and 384-well plates. The pipetting tasks to be performed in Practical class 1 are designed to
provide a check of the pipettes (and to refresh your pipetting skills), using an experimental design
very similar to that used in the later classes. The steps in the procedure should be performed in
pairs.
Procedure – Students
A. PREPARATION OF THE 96-well DYE STOCK PLATE: Prepare the dye stock plate (person 1)
Pipette 180 μL lots of each solution (A-H) into the corresponding wells of Column-1 of the
dye stock plate.
B. PREPARATION OF THE 96-well MIXING PLATE (person 2):
• Add 180 μL lots of Millipore water using an 8-channel pipette
• Remove the water by flicking the plate over the sink
• Add 180 μL lots of Millipore water using an 8-channel pipette
C. DILUTION OF DYE STOCKS IN THE MIXING PLATE (person 2):
• Add 20 μL lots of the dye stocks from the DYE PLATE to the MIXING PLATE using the 8-
channel pipette – 4 dispenses into Columns 1-4 (discard the pipette tips after each addition)
• Tap gently to mix.
• Read the plate at 428 nm using one of the three Absorbance plate readers
• Consult your demonstrator to determine whether the data are sufficiently reliable to justify
moving on to the next step (D)
D. MEASUREMENT PLATE (384 wells): Measure absorbances of diluted dye stocks (person 1)
• Place 5 μL lots of the samples from A1: H4 into the 384-well plate using an 8-channel
pipette
• Read the plate at 428 nm using one of the three Absorbance plate readers.
DYE STOCK PLATE (96-wells)
1 2 3 4 5 6 7 8 9 10 11 12
A Sol. A
B Sol. B
C Sol. C
D Sol. D
E Sol. E
F Sol. F
G Sol. G
H Sol. H
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 11
96-well MIXING PLATE (180 μL water + 20 μL dye stocks) – Plate with lid –
1 2 3 4 5 6 7 8 9 10 11 12
A Sol. A Sol. A Sol. A Sol. A
B Sol. B Sol. B Sol. B Sol. B
C Sol. C Sol. C Sol. C Sol. C
D Sol. D Sol. D Sol. D Sol. D
E Sol. E Sol. E Sol. E Sol. E
F Sol. F Sol. F Sol. F Sol. F
G Sol. G Sol. G Sol. G Sol. G
H Sol. H Sol. H Sol. H Sol. H
384-well MEASUREMENT PLATE (5 μL diluted dye solutions)
NB. There are 16 rows in a 384-well plate (vs 8 rows in the 96-well plates above). Thus, when you
transfer liquid from the 96-well plate using an 8-channel pipettor, this liquid will be dispensed
into every second well in the 384-well plate.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 12
Practical class 2: Effect of calcium on CaSR
stimulated phosphorylation of ERK1/2
General outline
The protein kinase ERK1/2 is phosphorylated in response to various stimuli including changes in
calcium levels. Phosphorylated ERK1/2 drives the expression of a large number of genes involved
in various processes (growth, development and memory formation etc). Measurement of ERK1/2
phosphorylation will be determined using an antibody-based assay (AlphaScreen).
There are three main steps in the experiment:
I. 3-hr serum starvation of cells
II. treatment of cells with different activators
III. measurement of ERK1/2 phosphorylation.
Students will work in groups of 3.
During the incubations, students can work on the GraphPad Prism Exercise (page 22).
After the practial, the demonstrators will collate the data, which will then be available on Canvas.
Please note that all handling of cells must be done inside the Laminar Flow (Class II Biosafety)
Cabinets within the X-lab, as indicated in the steps below.
Flow chart
Cultured HEK293 cells stably expressing the wild type CaSR (wtCaSR) will be exposed to Ca2+
concentrations between 0.5 mM to 8 mM. Phosphorylation of ERK1/2 will be measured as a
marker of CaSR activation.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 13
Cell Preparation (serum starvation) – Laboratory Staff
Materials and reagents
• Class II Biosafety Cabinet for tissue culture (T/C)
• T/C incubator with 5% CO2 set at 37°C
• Multi-well plate shakers
• 8-channel pipette with multi dispense capability
• ‘Repettors’ to repetitively deliver up to 6 μL lots into individual wells
• Sterile 1250 μL tips
• Calcium free DMEM that contains 0.2% (2 mg/mL) BSA
• 30% BSA – sterile tissue culture grade (Sigma)
• Physiological Saline Solution (PSS) with 0.2 mM Ca2+ and 0.2% (i.e., 2 mg/mL) BSA
• Sterile 1M CaCl2 stock
• Components required to make the ERK1/2 Detection Solution (scale this up to be suitable for
25 plates; each plate requires a minimum 224 μL -> 250 μL; 6.25 mL):
(i) Reaction Buffer 570 μL (*8.65 = 4.9 mL)
(ii) Activation Buffer 140 μL (*8.65 = 1.2 mL)
(iii) Orange Bead Stock 8 μL (*8.65 = 0.42 mL)
(iv) Blue Bead Stock 5 μL (*8.65 = 43 μL)
Procedure (prepare in sterile conditions in tissue culture hood)
• Prepare DMEM with 1.5 mM CaCl2 and 0.2% (2 mg/mL) BSA
• Remove complete DMEM from the cell-containing plates by flicking the plates
• Add 150 μL of DMEM with calcium & BSA
• Incubate for 3 h in T/C incubator (37°C)
Student Procedures
Materials and reagents
• Two incubators: Cell-Culture Incubator (5% CO2) and non-CO2 incubator, both set at 37°C
• 96 well poly-D-lysine coated plate for cell culture: 2 per group
• Physiological Saline Solution (PSS) with 0.2 mM Ca2+ and 0.2% BSA (i.e., 2 mg/mL)
• (125 mM NaCl, 4 mM KCl, 20 mM HEPES, 0.1% D-glucose, 1 mM MgCl2, 0.2 mM CaCl2)
• 8-channel pipette(s) that can dispense 5 μL, 20 μL & 160 μL (yellow tips)
• CaCl2 stock solution: 1 M CaCl2 in Millipore water (dilute as required in PSS to generate 10X
stocks: 5-80 mM to provide final concentrations of 0.5-8.0 mM)
• Lysis buffer diluted 1:5 from the ERK1/2 kit stock and kept on ice.
• 384-well PerkinElmer plates
• Multichannel pipette with multiple dispensing capabilities 50-1200 μL (for cell plating and
buffer addition) & 10-100 μL (drug addition and transfer of cell lysate): 1 per group
• Multipette (‘Repettor’) with 100 μL combi-tips to dispense 6 μL volumes
• Rooms with reduced light to add light sensitive beads to the plate
• BMG Clariostar plate reader
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 14
Procedure
A. DRUG PLATE: Prepare the drug plate (person 1)
Pipette 180μl lots of each solution (A-H) into the corresponding wells of Column-1 of the
drug plate.
B. CELL PLATE: Calcium starvation of cells (persons 2 & 3)
In the biosafety cabinet:
• Remove the media (pink solution) by flicking the plate over the plastic trough.
• Add 180 μL PSS solution using an 8-channel pipette and mix by gently shaking the plate
• Remove the PSS by flicking the plate over the plastic trough.
• Add 180 μL PSS using 8-channel pipette and incubate at 37°C (in the incubator) for 30 min
• Pour the contents of the trough into a Schott bottle (to be decontaminated with F10SC) and
decontaminate the biosafety cabinet with 80% (v/v) ethanol.
C. CELL PLATE: Cell stimulation with activator (calcium)
In the biosafety cabinet:
• Add 20 μL lots of calcium stock solutions from the DRUG PLATE to the CELL PLATE
containing the cells using the 8-channel pipette – 4 dispenses into columns 1-4 (discard the
pipette tips after each addition)
• Final extracellular Ca2+ concentrations (in mM) are: 0.5; 1.0; 1.5; 2.0; 3.0; 4.0; 6.0 and 8.0.
• Gently tap to mix the plate.
• Incubate at 37°C (in the non-CO2 incubator) for 5 min.
• Remove the PSS by flicking the plate over the plastic trough..
• Add 75 μL lysis buffer using 8-channel pipette.
• Take the plate out of the biosafety cabinet. Transfer the plate to an orbital shaker (low) to
provide gentle shaking of the cell lysate. Wait 15 min before the next step.
• Pour the contents of the trough into a Schott bottle (to be decontaminated with F10SC) and
decontaminate the biosafety cabinet with 80% (v/v) ethanol.
D. MEASUREMENT PLATE (384 wells): Measure phospho-ERK1/2 levels
• Place 5 μL lots of cell lysates into 384-well plate using the 8-channel pipette
• Under dimmed light conditions and using the multipette add 6 μL bead mix to the wells
containing 5 μL cell lysate in 384-well plate
• Seal the 384-well plate with a microplate cover to prevent evaporation.
• Cover the 384-well plate with aluminium foil and incubate in the dark for 2 h (room temp).
• Read the plate using the AlphaScreen plate reader – BMG Clariostar
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 15
96-well DRUG PLATE – flat bottom (180 μL)
1 2 3 4 5 6 7 8 9 10 11 12
A Sol. A
B Sol. B
C Sol. C
D Sol. D
E Sol. E
F Sol. F
G Sol. G
H Sol. H
96-well CELL PLATE (180 μL PSS + 20 μL drug) – Plate with lid – IMPORTANT PLATE
1 2 3 4 5 6 7 8 9 10 11 12
A Sol. A Sol. A Sol. A Sol. A
B Sol. B Sol. B Sol. B Sol. B
C Sol. C Sol. C Sol. C Sol. C
D Sol. D Sol. D Sol. D Sol. D
E Sol. E Sol. E Sol. E Sol. E
F Sol. F Sol. F Sol. F Sol. F
G Sol. G Sol. G Sol. G Sol. G
H Sol. H Sol. H Sol. H Sol. H
384-well MEASUREMENT PLATE (5 μL cell lysate + 5 μL Orange bead mix + 2 μL Blue bead mix)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 16
Practical class 3: Effect of calcium,
phenylalanine and cinacalcet on (1) wild-type
CaSR (wtCaSR) or 2) mutant (S169T) CaSR
stimulated ERK1/2 phosphorylation
General outline
Cultured HEK293 cells stably expressing:
(1) the wtCaSR OR
(2) mutant CaSR (S169T)
will be exposed to Ca2+ concentrations between 0.5 mM to 8 mM in the absence or presence of LPhenylalanine
(10 mM) or cinacalcet (0.3 μM). Phosphorylation of ERK1/2 will be measured as a
marker of CaSR activation.
Each group (3 students) will receive 1) a plate of cells that express wtCaSR AND/OR S169T CaSR, and
2) one drug (cinacalcet or L-Phe). Data will be pooled from the entire class for the analysis.
During the incubations, students can analyse their data from Week 2 and work on the “How to ttest”
exercise (available on Canvas).
After the practial, the demonstrators will collate the data, which will then be available on Canvas.
Please note that all handling of cells must be done inside the Laminar Flow (Class II Biosafety)
Cabinets within the X-lab, as indicated in the steps below.
Flow chart
Materials and reagents
The reagents and equipment used this week are identical to the second practical class, with the
inclusion of a ‘drug’ this week.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 17
Procedure
Prepare drug plate (person 1)
Pipette out 200 μL lots of PSS alone as well as 200 μL lots of PSS that contains either ‘drug 1’
(cinacalcet) or ‘drug 2’ (L-Phe), together with calcium solutions (A-H) as shown in the Drug Plate
Layout.
Calcium starvation of cells (person 2 & 3) – to be conducted in the Biosafety Cabinet
1. Remove the media (pink solution) by flicking the plate into waste container
2. Add 200 μL PSS solution and mix by gently shaking the plate using an 8-channel pipette
3. Remove the PSS by flicking the plate into waste container
4. Add 160 μL PSS using the 8-channel pipette and incubate at 37oC (in the incubator) for 30
min
5. Pour the contents of the trough into a Schott bottle (to be decontaminated with F10SC)
and decontaminate the biosafety cabinet with 80% (v/v) ethanol.
Cell stimulation with activators – to be conducted in the Biosafety Cabinet
1. Add 20 μL lots of PSS control as well as 20 μL lots of PSS containing drug from DRUG PLATE
to wells in CELL PLATE as shown in Plate Layout using the 8-channel pipette – 8 separate
horizontal solution dispenses are required from top to bottom (no need to change pipette
tips after each addition – OBSERVE THE PIPETTE TIP ORIENTATION, IF IN DOUBT, CHANGE
THE TIPS)
2. Incubate at 37oC (in the incubator) for 15 min
3. Add 20 μL of calcium stocks (solutions A-H) from the DRUG PLATE to the CELL PLATE
containing the cells using the 8-channel pipette – 4 separate vertical solution dispenses
from left to right (discard the pipette tips after each addition)
4. Gently tap to mix the plate.
5. Incubate at 37°C (in the non-CO2 incubator) for 5 min.
6. Remove the PSS by flicking the plate over the sink
7. Add 75 μL lysis buffer using 8-channel pipette (no need to change pipette tips after each
addition)
8. Transfer the plate out of the biosafety cabinet to an orbital shaker (low) to provide gentle
shaking of the cell lysate. Wait 15 min before the next step.
9. Pour the contents of the trough into a Schott bottle (to be decontaminated with F10SC)
and decontaminate the biosafety cabinet with 80% (v/v) ethanol.
Measure phospho-ERK1/2 levels
1. Place 5 μL of cell lysate into 384-well plate using the 8-channel pipette (discard the pipette
tips after each addition) – use columns 10-12 of white plate as shown in the plate layout
2. Gently mix the bead mix in the given tube by vortexing for 3-4 s.
3. Under dimmed light conditions add 6 μL bead mix to the wells containing the 5 μL cell
lysate in 384-well plate using the multipette (refill the multipette tip after every 16
dispenses i.e. 1 column)
4. Label your samples with your name and drug/phenotype!
5. Seal the 384-well plate with a microplate cover to prevent evaporation.
6. Cover the 384-well plate with aluminium foil and incubate at room temp in dark for 2 h.
7. Read the plate using the BMG Clariostar plate reader.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 18
Plate layout
96-well Drug Plate – flat bottom (275 μL) (A) PSS and Drug (1 or 2) and (B) Calcium sol. A-H
1 2 3 4 5 6 7 8 9 10 11 12
A PSS Drug PSS Drug Sol. A
B Sol. B
C Sol. C
D Sol. D
E Sol. E
F Sol. F
G Sol. G
H Sol. H
FIRST ADDITION (a) to 96-well Cell Plate (Add 160 μL PSS to all wells then add 20 μL PSS or Drug
from the Drug plate) – ADD TOP TO BOTTOM then pre-incubate for 15 min
1 2 3 4 5 6 7 8 9 10 11 12
A PSS Drug PSS Drug
B PSS Drug PSS Drug
C PSS Drug PSS Drug
D PSS Drug PSS Drug
E PSS Drug PSS Drug
F PSS Drug PSS Drug
G PSS Drug PSS Drug
H PSS Drug PSS Drug
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 19
SECOND ADDITION (b) to 96-well Cell Plate (To wells that already contain 180 μL PSS without or
with drug A/B now add 20 μL lots of the calcium solutions) – ADD LEFT TO RIGHT
1:PSS 2:Drug 3:PSS 4:Drug 5 6 7 8 9 10 11 12
A Sol. A Sol. A Sol. A Sol. A
B Sol. B Sol. B Sol. B Sol. B
C Sol. C Sol. C Sol. C Sol. C
D Sol. D Sol. D Sol. D Sol. D
E Sol. E Sol. E Sol. E Sol. E
F Sol. F Sol. F Sol. F Sol. F
G Sol. G Sol. G Sol. G Sol. G
H Sol. H Sol. H Sol. H Sol. H
384-well White Plate (5 μL cell lysate + 6 μL bead mix)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 20
Report assessment guidelines
Write a short scientific paper of no longer than two A4 pages of text with four sections as described
below.
1. Introduction:
a. what was the background to the experiments?
b. What were the aims of the experiments?
2. Methods:
Describe the key methods that were used.
3. Results:
What were your key results (support with at least two figures)?
4. Discussion:
Discuss the results in the context of the background information.
5. The figures and references should be provided in an Appendix on additional pages.
There is no page limit for the Appendix.
The marking scheme is provided on the next page, and additional details will be provided during the
Debrief Lecture.
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 21
NUTM3001 Nutrient Sensing Report – Marking Sheet
Please include your student ID in the file-name and front page of your report.
INTRODUCTION (/15)
Criteria Mark
What are GPCRs? Why are they important? 2
Why is regulating Ca2+ levels important? 2
CaSR – what does it do? 2
CaSR – how does it perform its function? What are its inputs (signals) and outputs
(effectors and responses)?
5
Aim(s) and hypothesis(es) of the experiments 4
With the exception of Aims/Hypotheses (which must be in the Intro), the other material can be in
either the Introduction or Discussion, so long as its location makes sense and you relate it to the
other things you are describing/discussing in that section.
MATERIALS AND METHODS (/5)
Criteria Mark
Methods explained clearly, including exceptions to the ‘published’ protocol. 2
References properly cited 1
Correct formatting (third person, past tense) 2
RESULTS (/30)
Criteria Mark
Week 2 data: Graphs/tables presented well, adequate legend(s), EC50 & n mentioned 5
Week 2 data: Trends interpreted properly and described in the text 5
Week 3 data: Graphs/tables presented well, adequate legend(s) 10
Week 3 data: Trends interpreted properly and described in the text 10
The data should include both the ERK phosphorylation data and the Hill equation results. See data
files for more information. See course manual about how to present graphs/tables.
DISCUSSION (/20)
Criteria Mark
Overview of the experiment(s). (What did you aim to do? What were your main
findings?)
5
Explain your results and how they compare to the literature (cite at least TWO papers) 5
The α-screen – what are the advantages/disadvantages of this assay? What other
assay could have been used instead?
5
What future experiments do you recommend? Propose at least TWO experiments
(remember to specify their aims/hypotheses).
5
What are the wider implications of this work? E.g. how does this relate to our
knowledge of CaSR and Ca2+ regulation, how does this relate to human physiology
and/or disease?
5
Yes, you can get 25/20 for this section. These items can be described in any order (preferably with
the overview first), as long as they’re all present in the Discussion.
REFERENCES (/10)
Criteria Mark
Reputable sources used to support statements?
At least two journal articles used?
5
References presented correctly and consistently? 5
Any referencing style is fine so long as it is consistent throughout the report (e.g. do not refer to an
article as [2] and then (Smith et al., 2009) elsewhere).
In addition to the general assessment guidelines in the course manual, please note:
• The total mark (out of 80) will be converted to a mark out of 10% for your final mark.
• The main report has a 2 page limit. Any text that exceeds this limit will not be marked, with the
exception of the Appendix (which has no limit).
• The minimum font size is 11. The minimum margins are 2cm. A penalty of 20% will be incurred
if these limits are exceeded.
• Marks will be capped at 80 (100%).
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 22
GraphPad Prism Exercise
Pre(r)amble
The optimal amount of time spent cramming for an exam is a topic frequently debated amongst
students. It is a well-known fact that prior to opening the textbooks, a certain amount of time must
be devoted to procrastination. Another commonly-accepted paradigm is the law of diminishing
returns, whereby spending extra time memorising minute details such as the textbook’s table of
contents will not make the High Distinction mark any Higher. However, a key aspect of this
controversy is caffeine usage in studying. For instance, coffee is known to dramatically improve
concentration, but excess amounts can lead to shaky hands, sleepless nights and metaphorical holes
in students’ wallets, all of which can impair academic success. Here, we investigate whether a
standard dose of caffeine alters the studying time required for a good mark.
Marks (/100)
Time
(h)
Study alone Study + Caffeine
Student 1 Student 2 Student 3 Student 1 Student 2 Student 3
0 2 2 0 3 2 1
2 5 6 8 8 7 9
4 12 15 14 28 25 30
6 29 27 29 51 48 57
8 51 45 43 65 68 74
10 64 59 61 79 82 86
12 81 72 76 92 95 93
14 91 85 92 97 98 99
16 96 95 96 100 100 97
18 98 95 99 98 98 100
20 100 98 97 100 99 99
What is the ‘X’ (independent variable)?
What is the ‘Y’ (dependent variable)?
NB. This data is completely fictitious.
Instructions (for Prism 7)
1. In the spreadsheet, you have been supplied with Grouped Lab Data for the effects of different
study times (in hours) on exam marks (/100) under (i) control conditions or in the presence of
(ii) caffeine.
2. Open GraphPad Prism.
3. Select ‘XY’ in the left column.
4. Under ‘Enter/Import data:’
a. ‘X’ (# hours): Numbers.
b. ‘Y’ (marks): Select second option. How many replicates do you need?
5. Press ‘Create’ in the bottom right hand corner
6. Copy the data from the Excel spreadsheet into Prism for (i) control, (ii) caffeine, ensuring the #
hours is in the X-column.
7. Save the Prism file at this point e.g., ‘Your initials-datafile1’
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 23
8. From the left hand menu, select ‘Graphs’ – Data 1 (the graph should be a collection of points
on an XY plot in which Time (h) is the x-axis and Marks is the y-axis)
9. Return to the data sheet. From the Top menu, under ‘Analysis’ tab select ‘Analyze’.
10. Under ‘XY’ analyses double-click ‘Non-linear regression (curvefit)’
11. Select ‘New’ in the top right hand corner.
12. Select ‘Create new Equation’ (you will now enter the Equation area)
13. Under ‘Name’ enter the text ‘Hill Equation’
14. Under ‘Definition’ enter the following text: Y=Bottom + (Top-Bottom)*X^n/(EC50^n+ X^n)
(Note for touchscreen keyboard only: to locate the ‘^’ symbol, Open the keyboard, Select the
keyboard tab, Select the third keyboard option from the left, Select ‘&1,2,3’ and then press ‘^’).
15. Once the equation is entered it saves automatically.
16. (Note for touchscreen keyboard only: Now close the keyboard).
17. Select the tab: ‘Rules for initial values’
18. Enter starting values for all four parameters using the keyboard (the optimization routine
requires starting values to calculate satisfactory estimates for ‘Bottom’ i.e., minimum mark,
‘Top’, i.e., maximum mark, ‘EC50’ i.e., the study time that results in a pass mark of 50, and the
Hill co-efficient ‘n’, which is a measure of the co-operativity between, say, different exam
questions). One satisfactory set of starting values for the four different parameters is as
follows: 1.0, 1.0, 1.0, 1.0. After entering the numbers, move between the cells without
attempting to save.
19. Finally, Press OK.
20. A page of results calculations should appear with the best-fit values for all four parameters.
Looking at the differences between minimum, maximum, EC50 and ‘n’, what are the effects
of caffeine on study efficiency? Which values would you present?
21. Copy these results to a new sheet of your Excel file and select ‘Save’.
22. Return to Prism. Select your Data Table, select ‘Analyze’ and double-click ‘Row means with SD
or SEM’ (under XY analyses) and then select ‘Row means with SEM’ and Select ‘OK’.
23. The graph should now appear showing the Standard Error of the Mean values (SEMs) for each
study time.
24. Clean up the graph to make it look more ‘publication-worthy’. For example:
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 24
Additional questions to think about
1. In Week 1, you subcultured cells. We also call this ‘splitting’ or ‘passaging’ (pronounced
‘passa(h)ging’ if you’re feeling posh).
a. You used trypsin/EDTA to release the cells. How does trypsin work?
b. Why doesn’t trypsin cleave itself?
c. How is it deactivated by adding serum-containing media?
d. James trypsinised some cells, and as soon as they had attached to the plate, he added Ca2+. He
found no ERK response. Why?
2. In this practical, you handled ‘HEK cells stably-expressing CaSR’.
a. What does this mean?
b. Why do we need to do this?
c. How could James use this to figure out which residues allow CaSR to interact with ERK?
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 25
Appendix – Preparation Room Procedures
I. Poly-D-Lysine coating of 96-well plates (Prep Room)
Requirements:
• Poly-D-Lysine (Sigma – 5mg bottle suitable for tissue culture)
• Sterile MQ
• Tissue culture hood
• 37°C incubator (DO NOT USE the one used to grow Bacterial culture)
• 8-channel pipette with multiple dispensing capability
• Sterile 1250 μL tips
• ‘Repettors’ to repetitively deliver up to 2 μL and 5 μL lots into individual wells
• Calcium free DMEM
• Sterile 1M CaCl2 stock
• 10 X concentrated CaCl2 stocks for use in the drug plate (5-80 mM)
• 30% BSA – sterile tissue culture grade (Sigma)
Protocol (sterile conditions)
1. Prepare a stock solution of poly-D-lysine in sterile MQ water – 200 μg/ml. The stock is stable
for up to a year if kept frozen
2. Dilute the above stock 8 times (5 mL stock + 35 mL sterile MQ) and add 50 μL of this to each
required well of 96-well plate
3. Incubate the plate at 37°C overnight
4. Next day empty the plate by flicking upside down
5. Rinse once with 100 μL sterile MQ and air dry for 20-30 min in the hood
6. Store the plates at 4°C until required. The plates are stable for 3 months
II. Cell plating (Prep Room)
Requirements:
• Tissue culture hood
• T/C incubator with 5% CO2 set at 37°C
• 8-channel pipette with multi dispense capability
• Sterile 1250 μL tips
• Poly-D-Lysine coated plates
• Complete DMEM media (DMEM + 10% FBS + pen/strep)
• 0.25% trypsin
• Haemocytometer
Protocol (sterile conditions)
1. Remove the Poly-D-Lysine coated plates from the fridge and bring them to room
temperature by putting them in the hood with the lid open to avoid water condensation.
2. Take a large (175 cm2) confluent flask of HEK-CaSR cells
3. Subculture the cells as per usual procedure
4. Count the cells using haemocytometer
5. Plate cells at a density of 60,000 cells per well in Poly-D-Lysine coated 96-well plates (6
columns)
6. Incubate overnight in T/C incubator
NUTM3001: Introductory Nutrition and Metabolism
Nutrient Sensing by G-Protein Coupled Receptors; Semester 1, 2020 26