Techniques

Cell culture multiple well, petri dish, timepoints, controls

Our Cellular Models, which cells in which experiments?

Protein analysis

‘quantitative’ (ELISA,RIA,EIA)

‘qualitative’ (immunocytochem, WIB, inmunoprecipitation)

EMSA

protein activity

Cytokine analysis

‘quantitative’

‘Activity” Functionality

mRNA analysis

Northern blot (quantification and stability)

RT-PCR (qualitative and quantitative)

Transfection assays with or without mutagenesis, deletion…

Cell function analysis

viability (MTT)

proliferation (3H incorp)

secretion (quantitative m/m of product)

oxidative stress (GSH, Hydrogen Peroxide, Lipoperoxidation assay, NO levels).

enzyme activity - AMPKinase (using Paranitrophenol as substrate)

cAMP measurement

Calcium

confocal, flu dyes, only in the literature

Electron Microscopy

fix, stain

Future EXTRACELLULAR COMUNICATION

Coculture ~ Primary- cell Lines

Coculture ~ Primary-Primary

Transcription factors

DNase I footprinting

EMSA / gel mobility assays

Cell cultureCell culture Primary cells (rat hepatocytes, Royal Free-human heps)Primary cells (rat hepatocytes, Royal Free-human heps)~5 day life, physiological functions, expression profile (R, ~5 day life, physiological functions, expression profile (R,

enzymes, proteins etc.)enzymes, proteins etc.) Transformed cells (human hepG2, HUH7, rat, mouse)Transformed cells (human hepG2, HUH7, rat, mouse)‘‘Infinite’ lifespan, loss of functions, expression profile Infinite’ lifespan, loss of functions, expression profile

Manipulation-stimulation (cAMP, DMSO), Manipulation-stimulation (cAMP, DMSO), -transfection (insertion of DNA), transgenic -transfection (insertion of DNA), transgenic rats/mice (deletion/insertion of DNA into rats/mice (deletion/insertion of DNA into animal), gene silencing (SiRNA)animal), gene silencing (SiRNA)

Culture conditions-plates (eg. 96 well plate:5x10Culture conditions-plates (eg. 96 well plate:5x1066-1x10-1x106 6

cells/well), cells/well), petri dishespetri dishes -medium, enriched (glucose, serum), -medium, enriched (glucose, serum), depleted depleted (steroids )(steroids )

The effects of cell culture The effects of cell culture on IGFBP profileon IGFBP profile

1 3 4 7 11

30

28

24

kDa

3 4 7 11 1 3 4 7 11day ofincubation

Large follicles Small follicles Large follicles

0

1 00

2 00

1 3 4 7 110

100

200

300

1 3 4 7 11 0

100

200

300

3 4 7 11

OD

Arb

itra

ry u

nit

s

30 kDa

28 kDa

24 kDa

day ofincubation

Fig. 9b(i)

Which cells in which experiments?Which cells in which experiments?

Cx expression and regulation Cx expression and regulation (protein/mRNA)(protein/mRNA)

Beta-adrenergic R effects (on cAMP)Beta-adrenergic R effects (on cAMP)

hepG2

HUH-7?

Primary

Fetal/non-proliferating/regenerating/transformed

Activity assaysActivity assays

E.g. enzyme. E.g. enzyme. – Provide substrate which changes optical Provide substrate which changes optical

properties (or is conjugated to something properties (or is conjugated to something which changes) e.g. MTT.which changes) e.g. MTT.

– Quantitative measurement of change in profile Quantitative measurement of change in profile of substrate by western blotting e.g. of substrate by western blotting e.g. intact/proteolysed, phosphorylated/non-Pintact/proteolysed, phosphorylated/non-P

E.g. hormone. E.g. hormone. – Measure biological consequence of activity e.g. Measure biological consequence of activity e.g.

insulin-stimulated glucose utilisation by cellsinsulin-stimulated glucose utilisation by cells

viability (MTT)viability (MTT)proliferation (proliferation (33H incorp)H incorp)

MTTMTTMitochondrial dehydrogenase converts MTT to dark Mitochondrial dehydrogenase converts MTT to dark

blue formazon (OD 570nm)blue formazon (OD 570nm)

Large intra-expt variability but can detect fold Large intra-expt variability but can detect fold changeschanges

33H incorporationH incorporationCells grown in labelled nucleotide which gets Cells grown in labelled nucleotide which gets

incorporated into DNA when proliferating. incorporated into DNA when proliferating. Radioactivity proportional to proliferationRadioactivity proportional to proliferation

More sensitive, lower variabilityMore sensitive, lower variability

GLUCOSE metabolism assaysGLUCOSE metabolism assaysTrends Endocrinol Metab. 1999 Dec;10(10):413-417. Trends Endocrinol Metab. 1999 Dec;10(10):413-417.    

Real-time Analysis of Glucose Metabolism by Microscopy.Real-time Analysis of Glucose Metabolism by Microscopy.

Piston DW, Knobel SM.Piston DW, Knobel SM.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, TN 37232, USA.702 Light Hall, Nashville, TN 37232, USA.

Glucose metabolism has traditionally been assayed via biochemical means. Glucose metabolism has traditionally been assayed via biochemical means. Fluorescence monitoring of NAD(P)H levels has provided a non-invasive Fluorescence monitoring of NAD(P)H levels has provided a non-invasive method to assay glucose metabolism in cells and tissues. However, these method to assay glucose metabolism in cells and tissues. However, these measurements have traditionally been of low resolution (no subcellular measurements have traditionally been of low resolution (no subcellular information) because of limitations imposed by optical and cellular information) because of limitations imposed by optical and cellular photodamage problems. The recent advent of two-photon excitation photodamage problems. The recent advent of two-photon excitation microscopy as a dependable tool for biological research has opened the microscopy as a dependable tool for biological research has opened the possibility of real-time, high-resolution analysis of glucose metabolism in possibility of real-time, high-resolution analysis of glucose metabolism in living cells. Such measurements have the potential to provide subcellular living cells. Such measurements have the potential to provide subcellular information from intact tissue that cannot be obtained by other techniquesinformation from intact tissue that cannot be obtained by other techniques

AMPKinase assay kitsAMPKinase assay kits(using Paranitrophenol/SAM as substrate)(using Paranitrophenol/SAM as substrate)

• Quantitative and qualitative

e.g.

Amersham cAMP assayAmersham cAMP assay

Enzyme immunoassay. Includes lysis reagents that eliminate lengthy sample extraction procedures and

allow direct cAMP measurement (optional use). Dual Range:  0.04-10.52 ng/ml, (0.125-32 pmol/ml)  [non-acetylation],  14-840 pg/ml,

 (40-2500 fmol/ml)  [acetylation]. Sensitivity:  38 pg/ml [non-acetylation],  14 pg/ml [acetylation].

5-h protocol. Store at 2-8 °C.

Qualitative Radio-immuno assay (RIA): Qualitative Radio-immuno assay (RIA): FSH-mediated steroidogenesis by follicular cellsFSH-mediated steroidogenesis by follicular cells

0

20

40

60

E2

fm

ol/1

000c

ells

/48h

t t+BP-4 t+FSH t+FSH+ BP-4

0

0.5

1.5

2.5

3.5

4.5

t t+FSH 0.5 5.0 50 BP-4 (ng/ml)

+ t + FSH

d

cbcb

a

a

t t+ BP-2 BP-3 BP-4 FSH t+ FSH+

0

0.5

1

1.5

2

2.5

3

a

b

c

E2

nm

ol/1

000

cell

s/48

h

Cells cultured +/- gonadotrophin+/- BP-2,-4 for 48h[Steroid] determined in the medium by RIA

Similar results for lactate production (as measured by an ELISA kit)

RT-PCRRT-PCR Qualitative-presence/absence and Qualitative-presence/absence and profileprofile (splicing, (splicing,

transcription start site variants) of mRNA for specific genetranscription start site variants) of mRNA for specific gene

mRNA isolated, reverse transcribed to cDNAmRNA isolated, reverse transcribed to cDNA

PCR performed on specific gene cDNAPCR performed on specific gene cDNA

Visualised on agarose gelVisualised on agarose gel

Quantitative modifications (TaqMan)Quantitative modifications (TaqMan)

Fluorescent probe added to PCR mix which fluoresces when Fluorescent probe added to PCR mix which fluoresces when incorporated into new DNA strand during PCR. Intensity of incorporated into new DNA strand during PCR. Intensity of fluorescence is proportional to original amount of cDNA. fluorescence is proportional to original amount of cDNA. However, quantity of original mRNA is calculated from ratio However, quantity of original mRNA is calculated from ratio with control gene mRNA levels in the same sample. Results with control gene mRNA levels in the same sample. Results between samples can then be compared.between samples can then be compared.

Fold differences can be measured but large inter-expt Fold differences can be measured but large inter-expt variation seenvariation seen

In vitroIn vitro functional studies for new polymorphisms: functional studies for new polymorphisms:Splicing profile of AAT mRNA transcripts in stimulated Splicing profile of AAT mRNA transcripts in stimulated

macrophages of different genotypesmacrophages of different genotypes

RT-PCR spanning intronsRT-PCR spanning introns

1A 1B 1C II III IV V1A 1B 1C II III IV V

5’5’ 3’3’

1A1B1C Both1A1B1C Both1A1C1A1C

• mRNA transcript mRNA transcript profile visualised on profile visualised on agarose gelagarose gel

Electron MicroscopyElectron Microscopy

QualitativeQualitative Field of view 20-0.5 cellsField of view 20-0.5 cells Only see electron-dense areas Only see electron-dense areas

(stain/fix…)(stain/fix…)

i.e. membranes, nucleii.e. membranes, nuclei

Functional analysis of novel polysFunctional analysis of novel polys Transplorer software: predicts Transplorer software: predicts

transcription factor binding sites transcription factor binding sites on DNA sequenceson DNA sequences

YY1 Oct 1CDP CR1 CDP CR3+HD

A allele G allele

Status of IGFBP-4 within Status of IGFBP-4 within follicular cellsfollicular cells

granulosa theca

Fig. 9a(i) IGFBP-4 WIB of GCCM (visualized by ECL)

lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

NHS

30

28

24

kDa

Regulation of IGFBP-4Regulation of IGFBP-4

0

5

10

15

20

25

30

Arb

itra

ry O

D u

nit

s

of 2

8KD

a p

rote

in

c t 10 1ng/ml

t+FSH

25%

48%

(ii) Mean optical density of 28kDa isoform of IGFBP-4

t+FSH t+FSH

28 kDa

NHS

Localisation of IGFBP-4 in Localisation of IGFBP-4 in follicular cellsfollicular cells

28 kDa(glycosylated,Intact)

16.5 kDa(fragment)

48 kDa(dimers)

CM AW N M C

LegendCM=conditioned mediumAW=cell surface associated fraction

N=nuclear fraction M=membrane fractionC=cytosolic fraction

Fig. 8a

Mechanism of IGFBP-4 effectsMechanism of IGFBP-4 effects

0

20

40

60

0

20

40

60

6h

3h

C LH LH+IGFBP-4

Pnmol/1000 cells/48h

(i)

a

b

a

b

c

c

t FSH FSH+ FSH+ FSH+ 8-br 8-br+ 8-br+ 8-br+ BP-4 BP-4 BP-4 BP-4 BP-4 BP-4 (50) (5) (0.5ng/ml) (50) (5) (0.5ng/ml)

Pn

mol

/100

0 ce

lls/

48h

0

15

30

45

a

d

c

b

c c

(i) GC (3-7mm follicles)

Timecourse

Effect on cAMP-mediated steroidogenesis

(i) (ii)

(iii) (iv)

(v) (vi)

5 m 5 m

5 m 5 m

10 m

20 m

(iv)(iii)

(ii)(i)

5 m

5 m 5 m

5 m

(v) (vi)

10 m

20 m