8 Anhang
8.1 Geräte und Chemikalien
8.1.3 Chemikalien , Reagenzien und Kits
Tabelle 15: Verwendete Chemikalien und Reagenzien
Produkt Name Konzentration Hersteller
Acrylamid Rotiphorese® Gel 30 Firma Carl Roth,
Karlsruhe, Deutschland
Aprotinin 10 mg/mL Firma Carl Roth,
Karlsruhe, Deutschland
APS Firma Carl Roth,
Karlsruhe, Deutschland Augensalbe Bepanthen
Augensalbe Bayer Vital GmbH,
Leverkusen, Deutschland
Benzamidin 10mg/mL Firma Carl Roth,
Karlsruhe, Deutschland
Beta-Glycerolphosphat 100mg/mL Firma Carl Roth,
Karlsruhe, Deutschland Bradford Reagenz
Biorad-Dye-Reagent-Concentrate Bio-Rad Laboratories,
Münschen, Deutschland
138
Tabelle 15: Verwendete Chemikalien und Reagenzien
Produkt Name Konzentration Hersteller
Bromphenolblau Firma Carl Roth,
Karlsruhe, Deutschland
Chemilumineszenz-Lösung Pierce®ECL Western
Blotting Substrate Pierce Biotechnologie, Rockford, USA
DAPI
Eindeckelmedium Vectashield Mounting
Medium Vector Laboratories,
Inc. Burlingame, CA, DSS Dextran-Sulfat- USA
Sodium-Salt-Reagent-Grade, Lot:
M5164, MP
MW:36000-50000 g/mol Biomedicals, Eschwege, Deutschland
DTT Biochrom AG, Berlin
Deutschland
FCS Low endotoxin Biochrom AG, Berlin
Deutschland
Formalin Formaldehyd 37% Firma Carl Roth,
Karlsruhe, Deutschland
Glycine Firma Carl Roth,
Karlsruhe, Deutschland
HEPES Pufferan Firma Carl Roth,
Karlsruhe, Deutschland
Isofluran Isofluran CP® CP-Pharma
Handelsgesellschaft mbH, Burgdorf, Deutschland
Klonidin Clonidine
hydrochloride Sigma Aldrich,
Steinheim, Deutschland
L-Glutamin 200 mM
low endotoxin Biochrom AG, Berlin Deutschland
139
Tabelle 15: Verwendete Chemikalien und Reagenzien
Produkt Name Konzentration Hersteller
Leupeptin 5mg/mL Firma Carl Roth,
Karlsruhe, Deutschland
Magermilchpulver Firma Carl Roth,
Karlsruhe, Deutschland Nicht-essentielle
Aminosäuren Sigma Aldrich,
Steinheim, Deutschland
Paraffin Paraffin Histoplast Thermo Fischer,
Walthan, USA Penicillin/Streptomycin 10000µg/mL
low endotoxin Biochrom AG, Berlin Deutschland
Pferde-Serum Oxoid Limited,
Hampshire, UK
Phenantroline 10mM Firma Carl Roth,
Karlsruhe, Deutschland
PMSF Fluka, Riedel-de Haën,
Seelze, Deutschland Proteinase K Proteinase K 50µg/µL Epicentre
Biotechnologies, Biozym Scientific GmbH, Oldendorf, Deutschland
Pyruvat Biochrom AG, Berlin
Deutschland Rinderserumalbumin Albumin Fraktion V ≥ 98%, MW
66000g/mol, Firma Roth, Karlsuhe, Deutschland
RPMI 1640 Medium Biochrom AG, Berlin
Deutschland
SDS Firma Roth, Karlsuhe,
Deutschland
Sodiumfluoride 1M (84mg in
2mL H2O) Firma Roth, Karlsuhe, Deutschland
Sodiumvanadat 100mM Firma Roth, Karlsuhe,
Deutschland SybrGreen Master-Mix Fast SYBR® Green
Master Mix Applied Biosystems,
Weiterstadt, Deutschland
140
Tabelle 15: Verwendete Chemikalien und Reagenzien
Produkt Name Konzentration Hersteller
Target retrieval
solution Dako, Hamburg,
Deutschland Taq-Man Master-Mix TaqMan® Fast
Advanced Master Mix Applied Biosystems, Weiterstadt,
Deutschland
TEMED Firma Roth, Karlsuhe,
Deutschland
Tris Firma Roth, Karlsuhe,
Deutschland
Triton-X SERVA
Electrophoresis GmbH, Heidelberg,
Deutschland
Trypsininhibitor 10mg/mL Firma Carl Roth,
Karlsruhe, Deutschland
Tween 20 Firma Roth, Karlsuhe,
Deutschland Zinksulfat Zink-Sulfat-Lösung 0,1M,
MW: 161,45 g/mol
Sigma Aldrich, Steinheim, Deutschland Tabelle 16: Verwendete Reaktionskits
Produkt Name Hersteller
Apoptose/TUNEL-Färbung In situ Cell Detection Kit, TMR red Roche, Basel, Schweiz DNA Verdau RNase freies DNase Set Quiagen, Hilden,
Deutschland
ELISA CD14 Quantikine®ELISA, Mouse CD14 R&D SystemsMinneapolis, Multiplex Kit mLn. USA
Kulturüberstand Magnetic Luminex® Screening
Assays R&D Systems GmbH,
Wiesbaden-Nordenstadt RNA Isolierung RNeasy Mini Kit Qiagen, Hilden,
Deutschland)
ReverseTranskriptase-PCR QuantiTect Reverse Transcription
Kit (Qiagen, Hilden,
Deutschland)
141 8.1.4 Puffer und Lösungen
4-Hydroxyethyl-1-Piperazinethansulfonsäure = HEPES Puffer (238,3012 g/mol) 0,5 M → 119 g Chemikalie für 1L Puffer, auf pH 7,4 einstellen
Phosphatgepufferte Salzlösung = PBS Rezept für 1L 10x PBS, auf pH 7,4 einstellen
Chemikalie Menge
NaCl 80,0 g (137 mM)
KCl 20,0 g (2,7 mM)
Na2HPO4 14,4 g (8,1 mM)
KH2PO4 2,4 g (1,5 mM)
Medium für Lymphozytenkultur
Zusätze für 500 mL RPMI-1640 Medium
Chemikalie Menge
Penicillin/Streptomycin 5 mL (1%)
L-Glutamin 5 mL (2mM)
Pyruvat 5 mL (1mM)
Nicht essentielle Aminosäuren
1x 5 mL
FCS 25 mL (5%)
Β-Mercaptoethanol 0,5 mL (50µM)
Lysepuffer für Proteinisolierung Rezept für 100 mL Pufferlösung
Chemikalie Menge
Tris HCl 1 M pH 7,4 2 mL
MgCl2 500 µL
EDTA 0,5 M 200 µL
EGTA 0,5 M 120 µL
142 Vor Versuchsbeginn, auf Eis gekühlt, dazugeben:
Aprotinin 1µL/mL Benzamidin 1,6µL/mL Leupeptin 2µL/mL Pepstatin 15µL/mL PMSF 20µL/mL Sodiumflaride 5µL/mL Β-Glycerolphosphat 22µL/mL Sodiumvanadat 1,5µL/mL Trypsininhibitor 1µL/mL Primathroline 5µL/mL
Zusammensetzung der Gele Rezept für jeweils 2 Gele
Chemikalie 12,5%-iges Trenngel 3%-iges Sammelgel
H2O 5,5 mL 6,3 mL
Tris-HCL pH 8,8 1,5 M 3 mL 2,5 mL Rotiphorese Gel 30 6,25 mL 1 mL
SDS 10% 150 µL 100 µL
APS 10% 75 µL 60 µL
TEMED 11 µL 11 µL
Ladepuffer für die SDS-Page Gelelektrophorese Rezept für 30 mL 5x Puffer
Chemikalie Menge
Glycerol 15 mL (50%)
SDS 3 g (5%)
Tris-HCL pH 8,8 0,5 M 7,5 mL (125 mM)
+ eine Spatelspitze Bromphenolblau
143
Elektrophorese-Puffer für die SDS-Page Gelelektrophorese Rezept für 1L 10x Puffer
Chemikalie Menge
Tris 30 g (250mM)
Glycine 144 g (1,92M)
SDS 10 g (1%)
Transfer-Puffer für den Western Blot Rezept für 5L Puffer, auf pH 8,3 einstellen
Chemikalie Menge
Tris 29 g (48mM)
Glycine 14,5 g (39mM)
SDS 1,85 g (0,037%)
Methanol 1 L (20%)
Wasch-Puffer für den Western Blot = TBS-Twen Rezept für 5L Puffer, auf pH 7,6 einstellen
Chemikalie Menge
Tris 12,1 g (20mM)
HCl 1M 19 mL (4mM)
NaCl 40 g (137mM)
Tween 20 5 mL (0,1%)
Block-Lösung für den Western Blot Rezept für 50mL Lösung
50mL TBS-Tween + 1,25 g BSA (2,5%)
+ 2,5 g Magermilchpulver (5%)
144 Stripping-Puffer für den Western Blot Rezept für 500mL Puffer, auf pH 2,2 einstellen
Chemikalie Menge
Glycine 7,5 g
SDS 0,5 g
Tween 20 5 mL
Erylyse-Lösung für die Transferkolitis
Rezept für 100mL 10x Lösung, auf pH 7,3 einstellen 100mL H2O
+ 8,99 g NH4Cl (1,7M) + 1 g KHCO3 (0,1M)
+ 37 mg EDTA-Na2-2H2O (1mM)
MACS-Puffer für die Transferkolitis Rezept für 50mL Puffer
50 mL PBs
+ 200 µL 5mM EDTA (2mM) + 0,25 g BSA
145 8.1.5 Antikörper
Tabelle 17: Verwendete Antikörper
Produkt Konzentration,
Klon Hersteller
AntiCD3: Zellkultur
purified anti-mouse CD3 0.5 mg/ml
Klon: 145-2C11 BioLegend, San Diego, USA AntiCD3: Zellsortierung
APC-Cy7 0,2mg/mL
Klon: 145-2C11 BioLegend, San Diego, USA AntiCD4: Zellsortierung
APC 0,2mg/mL
Klon: RM 4-5 BioLegend, San Diego, USA AntiCD8: Zellsortierung
PE-Cy7 0,2mg/mL
Klon: 53-6.7 BioLegend, San Diego, USA AntiCD14: WB
Goat IgG 0,1µg/ml
polyklonal R&D Systems, Wiesbaden, Deutschland
AntiCD45R: Zellsortierung
(B220)- VioBlue® 30µg/mL
Klon: RA3-6B2 Miltenyi Biotec GmbH,
Bergisch Gladbach, Deutschland
AntiCD62L: Zellsortierung
PE 30µg/mL
Klon:MEL14H2.100 Miltenyi Biotec GmbH,
Bergisch Gladbach, Deutschland
Anti GAPDH: WB
Goat 0,5 mg/mL
polyklonal GenScript USA Inc., NJ USA Anti Goat: Sekundär-AK
Donkey IgG antibody HRP 1mg/mL
polyklonal, antibodies-online GmbH, Aachen, Deutschalnd
Anti Ki67: Immunhistologie
rabbit 1 mg/mL
polyklonal Abcam, Cambridge, UK Anti Occludin:
rabbit 0.25 mg/mL
polyklonal Invitrogen, Darmstadt,
Deutschland Anti Rabitt: Sekundär-AK
donkey DyLight®594 0.5 mg/ml
polyklonal Abcam, Cambridge, UK
146 8.1.6 Primer
Tabelle 18: Verwendete Primer
Produkt Hersteller
Quantitect Primer assay Claudin 4
Mm_Cldn4_1_SG QuantiTect Primer Assay
Qiagen, Hilden, Deutschland Quantitect Primer assay Occludin
Mm_Ocln_1_SG QuantiTect Primer Assay Quantitect Primer assay Zonula-1
Mm_Tjp1_1_SG QuantiTect Primer Assay Quantitect Primer assay Beta-Aktin
Mm_Actb_2_SG QuantiTect Primer Assay Quantitect Primer assay CD14
Mm_CD14_SG QuantiTect Primer Assay TaqMan® Gene Expression Assay TNFα;
Mm00443258_m1
Applied Biosystems, Weiterstadt, Deutschland TaqMan® Gene Expression Assay IFNγ;
Mm01168134_m1
TaqMan® Gene Expression Assay Actb;
Mm00607939_s1
147
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