cell preparation and experimental lab dallas. cell lab – dallas
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Cell Preparation
and
Experimental Lab
Dallas
Cell Lab – Dallas
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
Zhen’s picture
The
Cells
111 billion
splenic B cells
A milestone
April 15, 2002 Vol. 1, No. 1 BCAfCS Research Reports www.afcs.org
Purification and Characterization of Mouse Splenic BLymphocytesScientific StaffAlliance for Cellular Signaling LaboratoriesUniversity of Texas Southwestern Medical Center, Dallas, TX and Veteran’s Administration Medical Center, San Francisco, CA
Abstract
Initial efforts of Alliance for Cellular Signaling(AfCS) laboratories have focused on establishing standardand reproducible procedures for purifying B lymphocytes(B cells) from mouse spleen. Our goal was to obtaincultures that permit reliable measurement of short-termsignaling events and changes in gene expression. Thisreport describes a negative-selection procedure for B cellenrichment and characterization of the isolated cellpopulation using multiparameter fluorescence-activatedcell sorting (FACS) analysis. Results show that we obtainapproximately 47 x 10 6 cells per spleen, of which 96%contain the B220 marker typically expressed on B cells.More than 80% of these cells can be further characterizedas resting, mature B2 cells. Conditions for maintainingviable cells in serum-free medium for short-term culturewere defined. Supplemented Iscove’s Modified Dulbecco’sMedium (SIMDM) was most consistent among severalmedia in maintaining cell viability for 24 hours. B cellsalso showed consistent and reproducible signaling
to manipulate gene expression in these cells based onreasonably complete knowledge of their genome. We alsowish to study relatively normal cells from a mammalianorganism. Based on these criteria one of the cells chosenwas the resting B lymphocyte (B cell) of the mouse.By secreting antigen-specific immunoglobulinantibody, the B cell plays an essential role in host defense.Resting B cells reside in the circulation and migrate to thespleen and lymph nodes. Activation of the B-cell receptorby antigen in conjunction with appropriate co-stimulationcauses the resting B cell to proliferate and differentiate intoa plasma cell, which makes and secretes large amounts ofimmunoglobulin antibody. In addition to the antigenreceptor, B cells exhibit a large number of additional cellsurface receptors, which are activated by T-cell surfacemolecules, cytokines, bacterial endotoxin, and otherligands. Immunologists have begun to define key steps insignaling pathways downstream of these receptors thatregulate steps in development of the mature B cell andmodulate responses to antigen (1,2,3).
Ligand Screen – B cells
Cell Lab in Dallas
Produces Cells
Treats Cellswith Ligands
Molecular Biology Lab
Microarray analysis
Protein Lab
P-proteins
Antibody Lab
P-proteins
Pilot project - Cornell
Lipid analysiscAMP
Calcium
Current Requirements
6 Outputs
Calcium
cAMP
RNA (microarray)
P-protein blots
2D gels
Lipids
B cells/assay (millions)
~1.5
1.5
18
18
18
6
Assays/year/6*
11,000
11,000
1000
1000
1000
3000
* Based on 100 billion B cells/year
Ligand Screen
Where are we?
Survey of the ligands
Ca2+ cAMP Immunoblot Conc Ligand
Yes NRAKT P90RSK ERK1
ERK250 mg/ml antigen/anti-Ig
NR NR AKT Stat6 6 ng/ml IL-4
NR NRAKT P90RSK ERK1
ERK210 mg/ml CD40L/CD154
YesMaybe (inhib?)
AKT P90RSK ERK1 ERK2
50 ng/ml SDF1/CXCL12
NR Yes NR 10 mM terbutaline
Yes NRP90RSK ERK1
ERK2200 ng/ml BLC/BCA-1/CXCL13
Yes NR AKT ERK1(?) TBD* SLC (CCL21; 6Ckine)
NR NR NR TBD IL-10NR NR NR TBD BAFF/BlyS (TNF family)
NRMaybe (inhib?)
NR TBD TRAIL
NR NR NR TBD IL-2NR NR AKT TBD DAMGO (?)NR NR NR TBD Dynorphin ANR NR TBD DADLE (?)NR NR NR TBD IL-5NR NR AKT P90RSK TBD IFNgNR NR AKT? TBD TNFaNR NR AKT? TBD IGF-1NR NR ERK1? TBD FasL*
TBD palmitoyl-lysoPCNR NR NR TBD Bombesin
NR Yes NR TBD 2-methyl-thio-ATP
NR NR NR TBD PAFNR NR NR TBD Carbachol
Table 1. Responses of Splenic B Lymphocytes to LigandsFacs Priority
Ly77 CD25 CD44 CD69 CD86
A
CD23 CD69 CD80 CD86
A
Ly77 CD18 CD23 CD25 CD44 CD54 CD69
CD86A
CD18(?) CD23 A
NR A
NR A
NR A
CD86? ACD23? A
NR A
NR BNR BNR BNR BNR B
MHC II? BNR B
Ly77? BNR B
BCD29? C
CD80 CD86 CD44 CD54 CD23
C
CCD80 CD44? C
Experiment DescriptionM7MH01112103.primary_lymphocytes_X00
Abs Call1
M7MH01112104.primary_lymphocytes_Y00
Abs Call2
Mean
AFFX-MURINE_B2_at K00131 mouse b2 repeat sequence from clone mm61 Control Sequence24282 P 22503 P 23393101054_at Ia-associated invariant chain 23426 P 22769 P 2309893086_at immunoglobulin kappa chain variable 28 (V28) 19592 P 17586 P 18589AFFX-b-ActinMur/M12481_3_at M12481 Mouse cytoplasmic beta-actin mRNA (_5 _M _3 represent transcript regions 5 prime Middle and 3 prime respectively) Control Sequence18931 P 17626 P 18279102156_f_at M80423:Mus castaneus IgK chain gene, C-region, 3 end /cds=(0,322) /gb=M80423 /gi=196865 /ug=Mm.46804 /len=323 mRNA18338 P 17575 P 17957102599_at translationally regulated transcript (21 kDa) 18039 P 17025 P 17532AFFX-b-ActinMur/M12481_M_atM12481 Mouse cytoplasmic beta-actin mRNA (_5 _M _3 represent transcript regions 5 prime Middle and 3 prime respectively) Control Sequence16793 P 15845 P 16319AFFX-b-ActinMur/M12481_5_at M12481 Mouse cytoplasmic beta-actin mRNA (_5 _M _3 represent transcript regions 5 prime Middle and 3 prime respectively) Control Sequence15693 P 15670 P 1568293583_s_at immunoglobulin heavy chain 6 (heavy chain of IgM) 15207 P 15323 P 1526597696_r_at ribosomal protein L7 15598 P 14410 P 1500494766_at eukaryotic translation elongation factor 1 alpha 1 14287 P 14794 P 14541100213_f_at ribosomal protein L41 15039 P 14013 P 1452693584_at immunoglobulin heavy chain 6 (heavy chain of IgM) 13410 P 13267 P 1333996358_at ribosomal protein S23 12592 P 11947 P 12270100780_at ribosomal protein S4, X-linked 12101 P 11279 P 1169093120_f_at histocompatibility 2, K region 10960 P 11581 P 11271100379_f_at AI837905:UI-M-AL0-abq-c-01-0-UI.s1 Mus musculus cDNA, 3 end /clone=UI-M-AL0-abq-c-01-0-UI /clone_end=3 /gb=AI837905 /gi=5472118 /ug=Mm.89082 /len=223 mRNA11920 P 10293 P 1110796300_f_at RIKEN cDNA 3200001M24 gene 11436 P 10342 P 10889101487_f_at Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, clone:0610010O05, full insert sequence10613 P 11088 P 1085195215_f_at ubiquitin C 10853 P 10604 P 1072998119_at ribosomal protein L30 11280 P 10148 P 1071496426_at thymosin, beta 4, X chromosome 11214 P 9873 P 10544100694_at ribosomal protein, large, P1 10718 P 10183 P 1045197647_at ribosomal protein S16 10769 P 9976 P 1037398085_f_at ribosomal protein S28 10841 P 9863 P 1035292866_at butyrophilin-like 2 10251 P 9555 P 9903102126_at ribosomal protein S12 10201 P 9342 P 9772101664_at ribosomal protein S3a 9959 P 9395 P 9677101255_at ubiquitin B 10272 P 8971 P 9622101869_s_at hemoglobin beta chain complex 9118 P 9124 P 9121100686_at ribosomal protein S2 9237 P 8971 P 9104100322_at expressed sequence AI893585 8993 P 8885 P 8939101577_at ribosomal protein S6 9552 P 8213 P 8883101573_f_at ribosomal protein L27a 9156 P 8439 P 879896290_f_at ribosomal protein L21 9010 P 8401 P 8706
Current Ligand List
LigandListB.xls
D:\LigandListB.xls
Priority Ligand Abb.
Receptor (IUPHAR name) Receptor (common name)
Standard ligands
A antigen/anti-Ig AIG BCR
A IL-4 I04 IL-4R
A CD40L/CD154 40L CD40
A SDF1/CXCL12 SDF 2.1.CXC.04.MMU.00.00CXCR4
A terbutaline TER 2.1.ADR.B2.MMU.00.00ß2AR
Ligand screen
A BLC/BCA-1/CXCL13 BLC 2.1.CXC.05.MMU.00.00CXCR5
A SLC (CCL21; 6Ckine) SLC 2.1.CC.07.MMU.00.00CCR7
A IL-10 I10 IL-10R
B IFNg IFG IFNgR
B TNFa TNF TNF-RII (and possibly R1)
B IGF-1 IGF
C 2-methyl-thio-ATP 2MA 2.1.NUCT.02.MMU.00.00 & 2.1.NUCT.05.MMU.00.00P2YR
C PGE2 PGE 2.1.PG.EP1.MMU.00.00 - 2.1.PG.EP4.MMU.00.00EP1R, EP2R, EP3R
C LTB4 (leukotriene B4) LB4 LTB4R
C NGF NGF
C Bombesin BOM
D fMLP FML
D Neurokinin B NEB
D Lysophosphatidic acid LPA Edg family (2,4,7)
D ELC (CCL19; MIP3b) ELC 2.1.CC.07.MMU.00.00CCR7
E Sphingosine-1-PhosphateS1P Edg family (1,3,5,6,8)
E CpG-oligodeoxynucleotideCPG Toll Receptor 9
C LPS LPS Toll Like Receptor-4 (or –2)
C TGF-beta TGF TGF-beta R I/II
E maxadilan MAX VIP1R
E RO-25-1553 R25 VIP2R
E CP55,940 C55 2.1.CBD.02.MMU.00.00Cannabinoid CB2
E CD27** 70L CD70
A BAFF/BlyS (TNF family) BAF U, (TNF-R family) (BMP, TACI)C PAF PAF PAFRD MIP3a M3A 2.1.CC.06.MMU.00.00CCR6
These were tested and showed a functional response.
These have not been offcially tested yet?
These were tested and gave no response but should be kept on list because of known
function(s) related to B cell biology.
Current Ligand List31 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
Patterns of Response
Group Ligand Calcium Cyclic AMP P-Protein Blots Genes Other
Gi S1P ++ 0 E/A/R SomeSDF ++ 0 E/A/R SomeBLC ++ 0 E/A/R SomeELC ++ 0 E/A/R SomeSLC ++ 0 E/A/R Some
Gs Terbutaline 0 ++++ (sust) 0 SomePGE2 0 ++ (sust) 0 Some2-MA (?) 0 + 0 Some
Gq & Gs LPA ++ (fast) ++ 0 Some
BCR Antigen ++++ 0 E/A/R Lots Proliferation
IL4 IL4 0 0 Stats 3 & 6 Lots
CD40 Anti-CD40 0 0 Delayed E/A/R Lots Proliferation
IL10 IL10 0 0 Stat 3 Yes
TNF TNFalpha 0 0 0 Yes
IGF IGF1 0 0 0 Yes
BAFF BAFF 0 0 0 Yes Viability
Assays:
cAMP
Challenges
EIA – competition of Peroxidase-cAMP with acetylated cAMP from samples
No PDE inhibitors
Rapid response
Assay interference
Solution
More sensitive assay – low fmol range switch to protocols using acetylation of cAMP
Effects of Propranolol on cAMP Responses
Time (min)
0 5 10 15 20
PropranololForskolinP+ F
Time (min)
0 5 10 15 20
cAM
P (
fmo
l/1x1
06 c
ells
)
0
50
100
150
200
250
300
PropranololTerbutalineP+ T
Terbutaline: 10 MPropranolol: 1 MForskolin: 100 M
10 M terbutaline 10 M PGE2
0.34 nM IL4 1 M LPA
cAMP – Ligand Interactionsc
AM
P (
fmo
l/mill
ion
ce
lls)
0
50
100
150
200
250
300
350
Co
ntr
ol
3 T
ER
Cont BLC ELC SDF SLC
cAM
P (
% o
f co
ntr
ol)
0
20
40
60
80
100
120
140
160+ 3 M terbutaline
Cont BLC ELC SDF SLC
cA
MP
(%
of
con
tro
l)
0
20
40
60
80
100
120
140
160No stimulation
O
N
O
N
COO-COO- -OOC
-OOCCa2+
O
N
O
N
COO-COO- -OOC
-OOCCa2+
EGTA
BAPTA
Fluo-3
O
N
O
N
-OOC-OOCCOO-
COO-
CH3
O O-O
Cl Cl
Ca2+
Calcium assay
• 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 – FminFree Ca++ = X Kd Fmax - F
Calcium response Value Std dev.
Peak response 129 nM 35 nM
Peak / baseline 2.03 0.35
Plateau response 82 nM 15 nM
Plateau / baseline 1.31 0.07
Time to peak 24 sec 5 sec
0.2 M BLC
1 mM LPA
Calcium response Value Std dev.
Peak response 130 nM 31 nM
Peak / baseline 1.71 0.07
Plateau response 96 nM 26 nM
Plateau / baseline 1.26 0.03
Time to peak 16 sec 2 sec
0
50
100
150
200
250
300
350
-200 -100 0 100 200 300 400 500 600
Time (sec)
[Ca2
+]
(nM
)
control
control
AIG
AIG
PGE,(10)
PGE,(10)
PGE(10)/AIG
PGE(10)/AIG
PGE(100)
PGE(100)
PGE(100)/AIG
PGE(100)/AIG
Interactions ?
0
50
100
150
200
250
300
350
-50 0 50 100 150 200 250 300
Time (sec)
[Ca2
+]
(nM
)
control
control
AIG
AIG
PGE,(10)
PGE,(10)
PGE(10)/AIG
PGE(10)/AIG
PGE(100)
PGE(100)
PGE(100)/AIG
PGE(100)/AIG
0
50
100
150
200
250
300
350
400
450
-50 0 50 100 150 200 250 300
Time (sec)
[Ca
2+]
(nM
)
control
control
AIG
AIG
TER(10uM)
TER(10uM)
TER(10uM)/AIG
TER(10uM)/AIG
TER(100uM)
TER(100uM)
TER(100uM)/AIG
TER(100uM)/AIG
3.8% 55.5% 86.0% 40.4%
cell
num
ber
cell
num
ber
cell
num
ber
cell
num
ber
Fluo-3 (fluorescence intensity)
Intracellular Calcium Response to Anti-IgM (0.3M)
Response of Ca: (0-5 min) stimulation with anti-IgM
Assays with
WEHI cells
0
200
400
600
800
1000
1200
-200 -100 0 100 200 300 400 500 600
Time (sec)
[Ca2+
] (n
M)
0
0
0.01
0.01
0.03
0.03
0.07
0.07
0.13
0.13
0.27
0.27
Calcium Assay in WEHI-231 CellsDose-dependent response to anti-IgM
0
1000
2000
3000
4000
5000
6000
7000
8000
0 5 10 15 20 25
Time (min)
cA
MP
(fm
ol/
1x
106
ce
lls
)
SIMDM
PGE2
TER
cAMP assay in WEHI-231 cells
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
CO-precipitation/mass spectrometry approach
• Express FLAG-tagged bait
• Immunoaffinity pulldown
• Gel separation
• Excised band/trypsin digest
• MS/MS
• Database search
Cell Lab
Expression of baits in WEHI cells
Retroviral Infection
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
Mscv-FTM-YFP
Mscv-neo-EGFP
MSCV-FTM-YFP
Quad Events % gated % totalUL 4.0 0.1 0.1UR 23.0 0.4 0.2LL 3758.0 57.8 37.6LR 2722.0 41.8 27.2
MSCV-NEO-EGFP
Quad Events % gated % totalUL 10.0 0.1 0.1UR 16.0 0.2 0.2LL 2199.0 31.7 22.0LR 4721.0 68.0 47.2
Retroviral Expression of Flourescent protein in WEHI
Mscv-neo-EGFP
MSCV-FTM-YFP
Quad Events % gated % totalUL 4.0 0.1 0.1UR 23.0 0.4 0.2LL 3758.0 57.8 37.6LR 2722.0 41.8 27.2
MSCV-NEO-EGFP
Quad Events % gated % totalUL 10.0 0.1 0.1UR 16.0 0.2 0.2LL 2199.0 31.7 22.0LR 4721.0 68.0 47.2
MSCV-FTM-YFP---CD4
How Long Can the Expression Last?
2 days
0
20
40
60
80
100
No Virus 24hr-1 24hr-2 24hr-3
%
8 weeks
0
20
40
60
80
100
No Virus 24hr-1 24hr-2 24hr-3
%
viability
transduced/living
transduced/total
Current state of retroviral infection
• 50-70% infection efficiency (105-106 cfu/ml)• >90% viability• Expression last over 8 wks
• Neomycin selection
Isolation of protein complexes
Pilot experiments with
CMV-3x-Flag-ERK2(from Melanie Cobb)
Transient expression in HEK293 cells
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
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
Lead Scientist
Zhen Yan
Technical Staff
Angela Alexander
Julie Collins
James Davis
Richard Davis
Jody Girouard
Joella Grossoehme
Read Pierce
Jason Polasek
Meghdad Rahdar
Associate Directors
Don Hilgemann
Richard Scheuermann
Scientific staff
Robert Hsueh
Keng-mean Lin
Yan Ni
The People
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