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Cell Preparation

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Cell Preparation and Experimental Lab Dallas Effects of Propranolol on cAMP Responses Calcium Assay in WEHI-231 Cells Dose-dependent response to anti-IgM CO ... – PowerPoint PPT presentation

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Title: Cell Preparation


1
Cell Preparation and Experimental Lab Dallas
2
Cell Lab Dallas
3
General Missions of Core C
Production of cells mouse splenic B cells
WEHI cells
mouse cardiac myocytes
Ligand Screen treatments
sample preparation
Assays for ligand screen cAMP
intracellular Ca
Protein traps cellular expression of baits
isolation of protein
complexes
4
Zhens picture
The Cells
5
A milestone
111 billion splenic B cells
6
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7
Ligand Screen B cells
Cell Lab in Dallas Produces Cells Treats
Cells with Ligands
Molecular Biology Lab Microarray analysis
Antibody Lab P-proteins
Pilot project - Cornell Lipid analysis
Protein Lab P-proteins
8
Current Requirements
B cells/assay (millions) 1.5 1.5 18 18 18 6
Assays/year/6 11,000 11,000 1000 1000
1000 3000
6 Outputs Calcium cAMP RNA (microarray) P-protei
n blots 2D gels Lipids
Based on 100 billion B cells/year
9
Ligand Screen Where are we?
10
Survey of the ligands
11
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12
Current Ligand List
LigandListB.xls
D\LigandListB.xls
13
Current Ligand List 31 approved ligands for the
initial B cell single-ligand screen yellow
tested and gave probable response (24)
blue not yet tested (4) tan no
response in original test but should be there (3)
4 additional ligands to be tried based on
expression in GNF arrays. 4 ligands - no
response in testing but still under re-evaluation
9 ligands - unresponsive in resting cells
probably due to absence of receptors, which are
expressed in response to stimuli (use for ligand
pairs?) 5 ligands have been rejected.
21 done in triplicate up to 18 more to
complete single ligand screen
14
Patterns of Response
15
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16
Assays cAMP
17
Challenges
No PDE inhibitors
Rapid response
Assay interference
Solution
More sensitive assay low fmol range switch
to protocols using acetylation of cAMP
EIA competition of Peroxidase-cAMP
with acetylated cAMP from samples
18
Effects of Propranolol on cAMP Responses
Terbutaline 10 mM Propranolol 1 mM Forskolin
100 mM
19
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20
cAMP Ligand Interactions
21
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22
Calcium assay
23
  • Load cells with dye
  • Incubate
  • Measure basal fluorescence
  • Add ligand measure fluorescence over time
  • Fmax use detergent to lyse cells and expose dye
    to saturating Ca to measure
  • Fmin - add EDTA to chelate Ca to measure basal
    fluorescent of dye

F Fmin Free Ca
X Kd Fmax -
F
24
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25
0.2 mM BLC
1 mM LPA
26
Interactions ?
27
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28
Intracellular Calcium Response to Anti-IgM (0.3?M)
cell number
cell number
cell number
cell number
Fluo-3 (fluorescence intensity)
29
Response of Ca (0-5 min) stimulation with
anti-IgM
30
Assays with WEHI cells
31
Calcium Assay in WEHI-231 CellsDose-dependent
response to anti-IgM
32
cAMP assay in WEHI-231 cells
33
Protein-interaction traps
2-hybrid in yeast Pull-downs with exogenous
tagged proteins Isolation of protein complexes
formed with tagged proteins expressed in
cells Co-immunoprecipitation of endogenous
complexes
34
CO-precipitation/mass spectrometry approach
Cell Lab
  • Express FLAG-tagged bait
  • Immunoaffinity pulldown
  • Gel separation
  • Excised band/trypsin digest
  • MS/MS
  • Database search

35
Expression of baits in WEHI cells
Retroviral Infection
36
  • General Strategy
  • Express tagged-protein driven by 5-LTR of virus
  • Bis-cistronic expression of a cell surface
    protein or antibiotic resistance gene for
    selection of cells expressing bait

37
Retroviral Expression of Flourescent protein in
WEHI
Mscv-FTM-YFP
MSCV-FTM-YFP---CD4
Mscv-neo-EGFP
38
How Long Can the Expression Last?
39
Current state of retroviral infection
  • 50-70 infection efficiency (105-106 cfu/ml)
  • gt90 viability
  • Expression last over 8 wks
  • Neomycin selection

40
Isolation of protein complexes
Pilot experiments with CMV-3x-Flag-ERK2 (from
Melanie Cobb) Transient expression in HEK293
cells
41
Prelimary Results Erk2 Associated Proteins in
293 cells
  • spectrin, alpha beta
  • ATP-dependent RNA helicase A
  • chromatin-specific transcription elongatiopn
    factor
  • splicing Factor 3b, subunit 1 2
  • ribosomal protein S6 kinase (Rsk2P90)
  • methylosome protein 50 (MEP50)
  • Human kinesin-related motor protein EG5
  • protein methyltransferase
  • rErk2
  • kiaa0122 gene product
  • Others

3x-Flag-Erk2
42
Goals of the next year
Ligand pair interaction in splenic B
cells (reduce material required)
Initiate screen in WEHI line
Initiate screen in cardiac myocytes ??
Optimize protein trap protocols - streamline
screens
Continue cooperative development of output assays
43
The People
Associate Directors Don Hilgemann Richard
Scheuermann
Technical Staff Angela Alexander Julie
Collins James Davis Richard Davis Jody
Girouard Joella Grossoehme Read Pierce
Jason Polasek Meghdad Rahdar
Lead Scientist Zhen Yan
Scientific staff Robert Hsueh Keng-mean Lin
Yan Ni
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