View
217
Download
0
Category
Preview:
Citation preview
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
Recommended