M ATERIALS

2.1.1DEVICES AND EQUIPMENT

TABLE 2.1DEVICES AND EQUIPMENT

DEVICE/EQUIPMENT MANUFACTURER

Agarose gel chamber Biofuge 15

CASY®-1 Cell counter Diavert microscope

Electrophoresis power supply

Emax microplate spectrophotometer Flow cytometer FACSCalibur^™

Flow cytometer/sorter FACSAria^™ III Gel photo-imaging system

Gel UV-imaging system Hera Cell 240 Incubator Hettich Rotanta Centrifuge Manual pipetting aid (Pipetus®) Micropipettes (10µl; 100µl; 1000µl) Multifuge 1 L-R

Skanwasher 400 Microplate Washer Thermocycler Eppendorf

Vortexer Reax 2000

OWL, Weilheim Heraeus, Hanau

Schärfe System, Reutlingen Leitz, Bielefeld

EC 150 – E-C Apparatus Cooperation American Laboratory Trading, USA Molecular Devices, Ismaning Becton Dickinson, Heidelberg Becton Dickinson, Heidelberg Mitsubishi, Ratingen

Fröbel, Lindau Heraeus, Hanau

Hettich Lab Technology, Tuttlingen Hirschmann, Eberstadt

Eppendorf, Hamburg Heraeus, Hanau

Molecular Devices, Ismaning Eppendorf, Hamburg

Heidolph, Schwabach

Used devices and equipment are listed with name and location of manufacturer.

55 2.1.2CHEMICALS

TABLE 2.2CHEMICALS

CHEMICAL MANUFACTURER

Ethanol

Ethidium bromide

Ethylenediaminetetraacetate (EDTA) H2O2

H2SO4

KCL KH2PO4

Isopropanol

NaCl

Na2HPO4 x 2H20

PBS ^def without Ca²⁺, Mg²⁺

Sodium azide (NaN3) Tween® 20

Ultra pure water

β-Mercaptoethanol 50 mM

Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Roth, Karlsruhe Biochrom AG, Berlin Roth, Karlsruhe Roth, Karlsruhe Biochrom AG, Berlin Gibco, Karlsruhe

Used chemicals listed with name and location of manufacturer.

2.1.3MEDIA AND SUPPLEMENTED MEDIA

TABLE 2.3MEDIA

MEDIUM MANUFACTURER

OptiMEM (with L-glutamine) RPMI 1640 (with Hepes)

Gibco, Karlsruhe PAA, Cölbe

Used media listed with name and location of manufacturer.

56 TABLE 2.4SUPPLEMENTED MEDIA

SUPPLEMENTED MEDIUM SUPPLEMENTS

OptiMEM^Suppl.

RPMI^Suppl.

Spleen DC isolation medium

500 ml OptiMEM (with L-Glutamine) 1 % FCS

100 U/ml Penicillin 100 µg/ml Streptomycin 0.05 mM β-Mercaptoethanol 500 ml RPMI 1640 (with Hepes) 10 % FCS

2 mM L-Glutamin 100 U/ml Penicillin 100 µg/ml Streptomycin 0.05 mM β-Mercaptoethanol 500 ml RPMI 1640 (with Hepes) 1 mg/ml Collagenase D

50 µg/ml DNase

Used supplemented media listed with supplements.

2.1.4MOLECULAR BIOLOGICAL REAGENTS

TABLE 2.5MOLECULAR BIOLOGICAL REAGENTS

REAGENT MANUFACTURER

6x LoadingDye

Agarose (Electrophoresis grade) Bovine serum albumin (BSA) Collagenase D

DNase

DOTAP Liposomal Transfection Reagent Dulbecco’s PBS

Fetal bovine serum (FBS) Flt3L

Generuler 1 kb DNA Ladder Plus

Fermentas, St. Leon Rot Invitrogen, Karlsruhe Roth, Karlsruhe Roche, Mannheim Roche, Mannheim Roth, Karlsruhe PAA, Cölbe Gibco, Karlsruhe

Cell culture supernatant, BMFZ Marburg

Fermentas, St. Leon Rot

57 Generuler 100 bp DNA Ladder Plus

GM-CSF GM-CSF

Lipofectamine 2000 Reagent L-glutamine 200 mM

LSM 1077 Separation Medium (Ficoll) M-CSF

o-Phenyl-Diamin (OPD) 20 mg tablet Penicillin/Streptamycin

Proteinase K

Recombinant murine IFN-α (Standard) Recombinant murine IL-6 (Standard) Streptavidin-POD

Taq PCR Master Mix Kit

Fermentas, St. Leon Rot PeproTech, Rocky Hill, USA Cell culture supernatant, BMFZ Marburg

Invitrogen, Karlsruhe PAA, Cölbe

PAA, Cölbe

PeproTech, Rocky Hill, USA Sigma-Aldrich, München PAA, Cölbe

Fermentas, St. Leon Rot HyCult Biotech, Uden, NL R&D Systems, Wiesbaden Roche, Mannheim

Qiagen, Hilden

Used molecular biological reagents listed with name and location of manufacturer.

2.1.5BUFFERS AND SOLUTIONS

TABLE 2.6BUFFERS AND SOLUTIONS

BUFFER/SOLUTION COMPONENTS

ELISA blocking buffer 1%

ELISA substrate buffer

ELISA washing buffer

10 g BSA

0.5 ml Tween® 20 Ad 1 L PBS def.

7.3 g C6H8O7

11,87 g Na2HPO4 x 2H2O Ad 1 L ddH20

500 ml 10x PBS 2.5 ml Tween® 20 Ad 5 L ddH2O

58 FACS buffer

FACS sorting buffer

PBS 10x

TAE DNA-gel running buffer 50x

Tail lysing buffer

PBS def.

2 % FCS 0.01 % NaN3

2 mM EDTA PBS def.

2 % FCS 2 mM EDTA 10 g KCl 10 g KH2PO4

400 g NaCl

57.5 g Na2HPO4 x 2H2O Ad 5 L ddH20

242 g Tris-Base

57.1 ml concentrated CH3COOH 100 ml 0.5 M EDTA (pH 8) 10 mM Tris HCL pH 8.0 25 mM EDTA

100 mM NaCl 0.5 % SDS (10 %)

Used buffers and solutions listed with required components.

2.1.6MOUSE STRAINS

TABLE 2.7MOUSE STRAINS

MOUSE STRAIN SOURCE

C57/Bl6 (wild type mice) HoxA9 knockout mice on C57/Bl6 background

Animal facility BMFZ Marburg Prof. Dr. Stefanie Dimmeler, Institute of Cardiovascular Regeneration, Goethe-University Frankfurt am Main

Used mouse strains and their source.

59 2.1.7ANTIBODIES

TABLE 2.8ELISAANTIBODIES

ELISAANTIBODY MANUFACTURER

Goat IgG biotinylated anti-mouse IL-6 PAb (Detection-Ab)

Goat POD-conjugated anti-rabbit IgG Mab (3.Ab)

Rabbit anti-mouse IFN-α PAb (Detection-Ab)

Rat IgG1 anti-mouse IFN-α MAb (Capture-Ab)

Rat IgG1 anti-mouse IL-6 MAb (Capture-Ab)

R&D Systems, Wiesbaden

Jackson ImmunoResearch, Suffolk, UK

PBL, Piscataway, NJ, USA PBL, Piscataway, NJ, USA R&D Systems, Wiesbaden

Used ELISA antibodies listed with name and location of manufacturer.

TABLE 2.9FACSANTIBODIES

FACSANTIBODY MANUFACTURER

ChromePure Rat IgG (Fc blocking) Rat anti-mouse BST2-FITC

Rat anti-mouse B220-APC Rat anti-mouse B220-FITC Rat anti-mouse CD11b-APC Rat anti-mouse CD11b-FITC Rat anti-mouse CD11c-APC Rat anti-mouse CD11c-PE Rat anti-mouse Flt3-PE Rat anti-mouse F4/80-FITC Rat anti-mouse Ly6C-PE

Jackson ImmunoResearch, Suffolk, UK

eBioscience, San Diego, USA BD Pharmingen, Heidelberg BD Pharmingen, Heidelberg eBioscience, San Diego, USA eBioscience, San Diego, USA eBioscience, San Diego, USA eBioscience, San Diego, USA BD Pharmingen, Heidelberg eBioscience, San Diego, USA Miltenyi Biotec, Bergisch Gladbach

60 Rat anti-mouse MHC-I-PE

Rat anti-mouse MHC-II-PE Rat anti-mouse SiglecH-FITC TO-PRO®-3 iodide

eBioscience, San Diego, USA eBioscience, San Diego, USA eBioscience, San Diego, USA Invitrogen, Karlsruhe

Used FACS antibodies listed with name and location of manufacturer.

2.1.8STIMULI

TABLE 2.10STIMULI

STIMULUS SEQUENCE 5’-3’/DESCRIPTION MANUFACTURER

LPS

ODN AP-1 PD ODN 1668 PD ODN 1720 PD ODN 2216 PS POLY I:C

RNA 40 PD R848

Pam-3-Cys

Lipopolysaccharide from Escherichia coli GCTTGATGACTCAGCCGGAA

TCCATGACGTTCCTGATGCT TCCATGAGCTTCCTGATGCT

GsGsGGGACGATCGTCGsGsGsGsGsGsG Polyinosinic-polycytidylic acid

GCCCGUCUGUUGUGUGACUC

Imidazoquinoline compound Resiquimod -selective synthetic ligand for murine TLR7 and human TLR7/8

Pam3CysSerLys4 – synthetic tripalmitoylated lipopeptide which activates TLR2

DIFCO, Detroit, USA

TIB MOLBIOL, Berlin

TIB MOLBIOL, Berlin

TIB MOLBIOL, Berlin

TIB MOLBIOL, Berlin

InvivoGen, San Diego USA GE Healthcare IBA Biologics, Göttingen InvivoGen, San Diego USA InvivoGen, San Diego USA

Used stimuli listed with sequence or description and name as well as location of manufacturer. ‘G’ – guanine; ‘C’ – cytosine; ‘A’ – adenine; ‘T’ – thymine; ‘U’ – uracil; ’s’ – phosphorothioate (PS) linkage.

61 2.1.9PRIMER

TABLE 2.11PRIMER

PRIMER SEQUENCE 5’-3’ MANUFACTURER

hoxa9-fp hoxa9-rp MC1neo-rp

CGCTGGAACTGGAGAAGGAGTTTCTG ATCCTGCGGTTCTGGAACCAGATC TCTATCGCCTTCTTGACGAGTTC

Metabion, Martinsried Metabion, Martinsried Metabion, Martinsried

Used primer listed with sequence and name and location of manufacturer. ‘G’ – guanine; ‘C’ – cytosine;

‘A’ – adenine; ‘T’ – thymine.

2.1.10CONSUMPTION ITEMS

TABLE 2.12CONSUMPTION ITEMS

ITEMS MANUFACTURER

24 G cannula

96-well ELISA MaxiSorp microplate 96-well flat bottom plate

96-well round bottom plate Cell culture dishes BD 10 cm

Cell culture dishes BD Primaria 10 cm Cell culture dishes Nunclon surface 10 cm Cell strainer 70 µm Nylon

FACS tubes sterile 5 ml snap cap (12 x 75 mm)

FACS tubes 1.3 ml (8.5 x 44 mm) Petri dishes 10cm

Pipette barrier tips (10 µl; 100 µl; 1000 µl)

BD Microlance, Heidelberg NUNC, Roskilde, Denmark Greiner, Frickenhausen Greiner, Frickenhausen BD Falcon, Heidelberg BD Falcon, Heidelberg NUNC, Roskilde, Denmark BD Falcon, Heidelberg

Fisher Scientific, Pittsburgh, USA BD Falcon, Heidelberg

Greiner, Frickenhausen Greiner, Frickenhausen

Sorenson, Salt Lake City, USA

62 Pipette tips (10 µl; 100 µl; 1000µl)

Pipette tips for multipette® (25 µl; 50 µl; 250 µl)

Plastic pipettes (5 ml; 10 ml; 25 ml) Reaction tubes (0.5 ml; 1.5 ml; 2 ml) Reaction tubes (15 ml; 50 ml)

Syringe 10 ml

Topseal A for 96-well microplates

Greiner, Frickenhausen Eppendorf, Hamburg Greiner, Frickenhausen Sarstedt, Nümbrecht Greiner, Frickenhausen B. Braun, Melsungen

PerkinElmer, Zaventem, Belgium

Used consumption items listed with name and location of manufacturer.

2.1.11SOFTWARE

TABLE 2.13SOFTWARE

SOFTWARE MANUFACTURER

BD FACSDiva^TM 7.0 (for Windows)

CellQuest Pro, version 5.2.1 (for Macintosh)

EndNote Web

FlowJo, version 10.0.7 (for Windows)

Microsoft Office 2007, 2010, and 2013 GraphPad Prism 6.05

SigmaPlot 10.0 SoftMax Pro V5

BD Bioscience, San Jose, USA BD Bioscience, San Jose, USA Thomson Reuters, Carlsbad, USA Treestar, San Carlos, USA

Microsoft, Redmond, USA

GraphPad Software, La Jolla, USA Scientific Solutions, Lausanne, Swiss Molecular Devices, Ismaning

Used software listed with name and location of manufacturer.

63

Im Dokument Impact of the Transcription Factor HoxA9 on Toll-like Receptor-Mediated Innate Immune Responses and Development of Dendritic Cells and Macrophages (Seite 80-89)