Core D, San Francisco: Laboratory for Development of Signaling Assays B Lymphocytes –Initiate...
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Transcript of Core D, San Francisco: Laboratory for Development of Signaling Assays B Lymphocytes –Initiate...
Core D, San Francisco: Laboratory for Development of Signaling
Assays
• B Lymphocytes– Initiate ligand screen, 1st publication (with Core C, Dallas)
– Long term culture
• Myocytes
Luyi Li
Tamara Roach
TimO’Connell
Paul Simpson Bill
Seaman
Melissa Kachura
SusanRicker
The SF VAMCAfCS Lab
Hanging Heart
• needle inserted in LV apex in situ• drain atrium & clamp aorta• constant pressure (~75 mmHg, 125 cm) or• constant flow (4 ml/mim)
In Situ Perfusion
pump
• dissect heart• cannulate aorta• constant flow (4 ml/min)
Constant Pressure
Steps in both:• Ca++ wash out• Collagenase digestion ( 50 M Ca++)• Mechanical disaggregation• Collagenase inhibition (BCS)• Ca++ reintroduction• Wash & count
pump
Constant Flow or Constant Flow
Myocyte Isolation Procedure
Hang. Heart In Situ(Const. Flow)
(50)Const. Pres.
(64)Const. Flow
(38)
Myocytes For Plating (106)% Rod ShapedRod Shaped Myocytes
2.6 ± 0.5 76 ± 10%
2.0 ± 0.5
1.6 ± 0.3 68 ± 8%1.1 ± 0.3
1.7 ± 0.3 67 ± 7%1.1 ± 0.3
# 35 mm Dishes at 50K rods/dish(~62 rods/mm2)
39 22 22
Plating Efficiency@ 1 hr (%)(#attached/#plated)
39%*
In Situ preparation is much easier technicallyIn Situ constant flow preparation is easier than constant pressure
* measured since January, 2002
Myocyte Yields with DifferentIsolation Techniques
0 hr 24 hr 72 hr
Plate for 1 hr on laminin coated dishes in:MEM w/Hanks BSS w/5% BCS10 mM BDMPenicillin
Change Medium to:MEM w/Hanks BSS w/1 g/ml Insulin0.5 g/ml Transferrin0.55 ng/ml Selenium1 mg/ml BSA10 mM BDMPenicillin
Culture for up to 72 hours at 37°C in 2% CO2
Goal: Maintain rod-shaped myocytes that signal for 72 hrs
Myocyte Culture Procedure
MyocyteIsolation
3 hrs
PlatingAssay
SignalingAt 24 hrs
AssaySignalingAt 72 hrs
1 hr 24 hrs 48 hrs
MediumChange
Assays:Gs: cAMP, PLB phosphorylation, myocyte contractionGi: inhibition of cAMPGq: ERK phosphorylation
Myocyte Experimental Timeline
10-10 10-9 10 -8 10 -7 10-6 10-5
20000
8000
12000
16000
4000
0
Isoproterenol (nM)
fmo
l cA
MP
/2
0,0
00
myo
cyt
es
Isoproterenol, a -AR agonist that signal through Gs,increases cAMP in a concentration-dependent manner
EC50 24 nM72 hrs
EC50 28 nM
24 hrs
Activation of Gs Signaling in Myocytes at 24 and 72 Hours
Control Iso
120000
100000
800003500030000
200001500010000
05000
25000
Fsk FskCarb
IsoCarb
Isoproterenol (1 M)Forskolin (100 M)Carbachol (100 M)
fmo
l cA
MP
/2
0,0
00
myo
cyt
es
Carbachol, a muscarinic agonist that signals through Gi, reduces isoproterenol- and forskolin- induced cAMP accumulation
72 hrs
24 hrs
Activation of Gi Signaling in Myocytes at 24 and 72 Hours
Control PE20 M
ET-1100 nM
PMA100 nM
Phospho-ERK
Total-ERK
Phenylephrine, an 1-AR agonist, and Endothelin-1 which both signal through Gq, increase ERK1/2 phosphorylation
Control PE20 M
ET-1100 nM
PMA100 nM
24 hrs 72 hrs
Activation of Gq Signaling in Myocytes at 24 and 72 Hours
Control Iso1 M
Phospho-PLB
G
Isoproterenol, a -AR agonist that signals through Gs,increases phospholamban phosphorylation
Control Iso1 M
24 hrs 72 hrs
Phospholamban Phosphorylation in Myocytes at 24 and 72 Hours
QuickTime™ and a decompressor
are needed to see this picture.
QuickTime™ and a decompressor
are needed to see this picture.
Activation of E-C Coupling in Myocytes at 24 Hours
Myocytes contracting under field stimulationMyocytes quiescent for first 5 secondsStimulated at 80V, 1 Hz for 20 seconds
Then increase frequency to 1.5 Hz for 15 seconds
QuickTime™ and a decompressor
are needed to see this picture.
QuickTime™ and a decompressor
are needed to see this picture.
Isoproterenol, a -AR agonist that signals through Gs and increases phospholamban phosphorylation,
induces myocyte contraction
Activation of E-C Coupling in Myocytes at 24 Hours
Control 1 M Isoproterenol
0
2
4
6
8
% S
ho
rte
nin
gIsoproterenol induces myocyte contraction
16 16
Myocyte Contraction measured as %Shortening of individual cardiac myocytes
*
* p < 0.05
Activation of E-C Coupling in Myocytes at 72 Hours
270%
Assay Time Points MyocytesPhosphoprotein 0, 2, 5, 12, 30 min 5 x 35 mm dish
(250,000 myocytes)
RNA Array 0, 30, 120, 240 min 16 x 60 mm dish
(2,400,000 myocytes)
cAMP 0, 2, 5, 12, 30 min 5 x 35 mm dish
(250,000 myocytes)
Calcium in development
Total 2.9 x106 myocytes
Project that we need 3 hearts/ligand
Requirements for the Ligand Screen
Summary: Myocytes
• Criteria– Acute signaling: cAMP,
phosphorylation, contraction.– Suitable for mutation (RNAi, antisense,
transfection, etc).– Reproducible within and between labs.– Convenient, sufficient throughput.– Mouse, normal, adult.