PEST domain
III. Materials and Methods
1. Chemicals, Immunoreagents and Disposable Goods
Table 5: List of antibodies applied for flow cytometry and confocal microscopy
Antigen clones Provider Label
CD14 MfP9 Becton-Dickinson, Heidelberg,
Germany APC
CD45 2D1 Becton-Dickinson, Heidelberg, Germany
PerCP Ceramide MID15B4 Alexis, Grünberg, Germany Goat anti
mouse-FITC CDw17 G035 Becton-Dickinson, Heidelberg,
Germany
FITC CD65s VIM2 Caltag, Burlingham, California,
USA
FITC CD77 38-13 Coulter/Immunotech, Krefeld,
Germany
Goat anti rat-PE
CD64 22 Coulter/Immunotech, Krefeld,
Germany
FITC
CD32 2E1 Coulter/Immunotech, Krefeld,
Germany
PE
CD16 3G8 Coulter/Immunotech, Krefeld,
Germany
FITC CD11a 25.3 Coulter/Immunotech, Krefeld,
Germany
FITC CD11b D12 Becton-Dickinson, Heidelberg,
Germany
PE
CD18 7E4 Coulter/Immunotech, Krefeld,
Germany
FITC CD36 FAG.152 Coulter/Immunotech, Krefeld,
Germany FITC
CD91
A2MR-a2
Becton-Dickinson, Heidelberg, Germany
FITC CD163 GHI/61 Becton-Dickinson, Heidelberg,
Germany
PE CD47 B6H12 Becton-Dickinson, Heidelberg,
Germany
FITC CD55 IA10 Becton-Dickinson, Heidelberg,
Germany
PE apoCI 10-F4 ICN Biomedicals, Eschwege,
Germany
Goat anti mouse-FITC apoE 21-F3-D2 Biotrend, Köln, Germany Goat anti
mouse-FITC
CETP ATM192 Dianova, Hamburg, Germany Goat anti
mouse-FITC Adipophilin AP125 Research Diagnostics,
Flanders, USA
Goat anti mouse-FITC Annexin V-Apoptosis
detection kit
Becton-Dickinson, Heidelberg, Germany
FITC
Antigen clones Provider Label Secondary antibodies
Goat F(ab)´2 anti mouse Ig Becton-Dickinson FITC
Goat F(ab)´2 anti rat IgM Dianova PE
Goat polyclonal to mouse IgG H&L
Abcam, Cambridge, UK Cy5
Goat polyclonal to anti-rabbit
Calbiochem, Schalbach, Germany
Texas Red Toxins
Cholera toxin subunit B (recombinant) (GM1)
Molecular Probes Europe BV, Leiden, Netherland
Alexa Fluor 488 Cholera toxin subunit B
(recombinant) (GM1)
Molecular Probes Europe BV, Leiden, Netherland
Alexa Fluor 555 Theta-Toxin
(perfringolysin O)
Biotechnology workgroup of the Center of Excellence for Fluorescent Bioanalysis (KFB),
Regensburg, Germany
FITC
Others DMPE-TMR (1,2-
dimyristoyl-sn-glycero-3- phospho-ethanolamine-tetramethyl-rhodamine)
Avanti Polar Lipids, Inc., Alabama, USA
POPC (1-palmitoyl-2-oleoyl-
sn-glycero-3-phosphocholine)
Avanti Polar Lipids, Inc., Alabama, USA
Lysenin gift from Sobota A, Nencki
Institute of Experimental Biology, Department of Cell
Biology, Warsaw, Poland anti-His probe (H-15) Santa Cruz Biotechnology, CA,
USA)
PE = Phycoerythrin, FITC = Fluorescein-isothiocyanate, PerCP = Peridin chlorophyll protein, APC = Allophycocyanin
Table 6: List of antibodies applied for western Blot analysis
Antibody clone provider host gel
ABCA1 AB.H10 Abcam, Cambridge, UK mouse
ApoE Biodesign, Germany goat
ApoC-I 10-F4 ICN Biomedicals,
Eschwege, Germany
mouse
PLTP kind gift from
Jauhiainen M., National Public Health Institute,
Department of Molecular Medicine,
Helsinki, Finland
mouse
ATP-synthase (b-chain)
3D5AB1 Acris, Hiddenhausen, Germany
mouse
CD14 Santa Cruz, CA, USA goat non-reducing
CD11b Santa Cruz, CA, USA goat
CD16 2H7 Serotec, Eching,
Germany
mouse non-reducing
CD47 BRIC126 Acris, Hiddenhausen, Germany
mouse non-reducing
CD55 Santa Cruz, CA, USA rabbit
CD163 EDHu-1 Serotec, Eching,
Germany
mouse
CD64 Santa Cruz, CA, USA goat
CD32 Santa Cruz, CA, USA goat
CD11a Santa Cruz, CA, USA goat
CD36 Santa Cruz, CA, USA rabbit
Medium, solutions and chemicals
Macrophage-SFM Medium, low endotoxin Invitrogen, Karlsruhe, Germany
M-CSF, human (rec. from E. Coli) R&D Systems, Minneapolis, Minnesota
PBS (w/o Ca2+, Mg2+) Gibco BRL, Berlin, Germany
BSA (lipid free) Sigma, Taufkirchen, Germany
FCS (Fetal calf serum) Gibco BRL, Berlin, Germany
Saponin Sigma, Taufkirchen, Germany
sodium azide Sigma, Taufkirchen, Germany
formaldehyd Sigma, Taufkirchen, Germany
CuSO4 Sigma, Taufkirchen, Germany
DMSO Sigma, Taufkirchen, Germany
SDS Sigma, Taufkirchen, Germany
Cholesterol esterase Roche, Basel, Switzerland
Trypsin SAA, Basel, Switzerland
Trizol Life Technologies, Karlsruhe, Germany
ALLN (N-acetyl-Leu-Leu-norleucinal) Calbiochem, Schalbach, Germany 1,1`-Dioctadecyl-3,3,3´,3´-tetramethylindo- Sigma, Taufkirchen, Germany carbocyanine-perchlorate (Dil)
9-cis-retinoic-acid Sigma, Taufkirchen, Germany
all-trans-retinoic acid
b-Carotene Sigma, Taufkirchen, Germany
Rosiglitazone Cayman Chemical, MI, USA
Ciglitazone Cayman Chemical, MI, USA
Troglitazone Cayman Chemical, MI, USA
Pioglitazone Cayman Chemical, MI, USA
human plasma apolipoprotein A-I and apoE Calbiochem, CA, USA
Kits
BCA, Protein Assay Kit Pierce, Rockford, IL, USA
ECL Western Blotting Analysis System Amersham, Braunschweig, Germany
Superscript II kit Life Technologies, Karlsruhe, Germany
RNeasy MiniKit Qiagen, Hilden, Germany
RNA 6000 Nano Chip Agilent, Palo Alta, CA, USA
Reverse Transcription System Promega, Madison, AL, USA Enzo BioArray HighYield RNA Enzo Life, Farmingdale, NY, USA Transcript Labeling Kit
Cy3, Cy5 Mono reactive dye pack Amersham, NJ, USA
PD-10 columns Amersham, NJ, USA
Microarray
Human Genome U133 Plus 2.0 Array Affymetrix, Santa Clara, CA, USA
Cell culture dishes
Ultra low attachment 6-well plates Costar Corning, Bodenheim, Germany LAB-TEK borosilicate chamber slides Nalge Nunc Int., Naprville, IL, USA
Falcon 6- well cell+ Becton Dickinson, NJ, USA
cell+ 10 cm tissue culture dishes Sarstedt, Inc. Newton, USA
Instruments
Heraeus 6000 Incubator Heraeus, Hanau, Germany
FACSCalibur flow cytometer Becton Dickinson, Heidelberg, Germany
2100 Bioanalyzer, Caliper Agilent, Palo Alto, CA, USA
ABI Prism 7900 HT Sequence Detection System Perkin Elmer-Applied Biosystems,
(Taqman) Darmstadt, Germany
TCS-4D confocal microscope Leica Lasertechnik, Omnichrom equipped with a ArKr 75 mV mixed ion laser Heidelberg, Germany Melles Griot, electrospray ionization tandem mass spectrometry
(ESI-MS/MS)
Thermocycler Gene Amp PCR System 9600 Perkin Elmer, Uberlingen, Germany GeneQuant pro RNA/DNA Calculator Amersham, Braunschweig,Germany Gene Chip Fluidics Station 450 Affymetrix, Santa Clara, CA, USA Gene Chip Scanner 3000 Affymetrix, Santa Clara, CA, USA Affymetrix array scanner Affymetrix, Santa Clara, CA, USA
ELISA reader Tecan, Stuttgart, Germany
Analysis Software
Cellquest software for FACS Analysis Becton Dickinson, Heidelberg,Germany Affymetrix Microarray Suite (version 5.0) Affymetrix, Santa Clara, CA, USA ScanWare software package Universal Imaging, Corp., PA, USA Metamorph for microscopy image Analysis Universal Imaging, Corp., PA, USA
Assays on Demand
gene-specific assays on demand were supplied by ABI.
Mastermix: 10 µl 2x TaqMan® Universal PCR Master Mix, 1 µl gene-specific probe-primer-mix and 4 µl sterile water
Mastermix for the endogenous control 18sRNA: 10 µl 2x TaqMan® Universal PCR Master Mix, 1 µl pre-developed TaqMan® assay reagents (PDAR) endogenous control kit and 4 µl sterile water
Table 7: Assays on demand
Assays on demand Assay ID
sphingomyelin synthase 1 Hs00153716
sphingomyelin synthase 2 Hs00380453
acid sphingomyelinase like phosphodiesterase Hs00205522 sphingomyelin phosphodiesterase 1 (acid SMase) Hs00609415 neutral sphingomyelinase (neutral SMase) activation associated factor Hs00182519 sphingomyelin phosphodiesterase 2 neutral SMase Hs00162006
CD36 Hs00169627
glucosidase, beta;acid (includes glucosylceramidase) Hs00400295 N-acylsphingosine amidohydrolase (acid ceramidase) 1 Hs00188842
UDP-glucose ceramide glucosyltransferase Hs00326535
sphingosine kinase 1 Hs00184211
sphingosine kinase 2 Hs00219999
serine palmitoyltransferase, long chain base subunit 1 Hs00272311 serine palmitoyltransferase, long chain base subunit 2 Hs00191585