2 Material and Methods
2.1.8 Buffers and Solutions
spleen cells were cultured in DMEM media supplemented with 20 % FCS, 1 % Penicillin/Streptomycin and 100 U/ml IL 6 at 37 °C and 5 % CO2 atmosphere.
2.1.6 Plasmids
Plasmid Manufacturer
pGEM®‐T Easy Promega
pAcGFP‐N1 Clontech
pFUSE‐hIgG‐Fc2 Invivogen
2.1.7 Size Standards
Size standard Manufacturer
GeneRuler 100bp ladder plus Fermentas PageRuler prestained protein ladder Fermentas
2.1.8 Buffers and Solutions
Buffer/Solution Preparation
BSA/Triton‐X‐100 buffer 500 mg BSA
350 mg Triton‐X‐100
adjust volume to 50 ml with HPLC‐H2O
ELISA ‐ Blocking solution 4 % milk powder in PBS
ELISA ‐ Coating buffer A: 0.2 M Na2CO3 B: 0.2 M NaHCO3
Mix 85 ml A with 40 ml B and adjust to 500 ml with H2O before use
ELISA ‐ Reaction buffer 0.1M NaAc
0.20.05 M NaH2PO4 pH 4.0
ELISA ‐ Substrate reaction 10 ml Reaction buffer 500 µl 40 mM ABTS 10 µl H2O2 (30 %)
Erythrocyte lysis buffer 8.29 g NH4Cl 1 g KHCO3
0.2 ml of 0.5 M EDTA adjust pH to 8.0
Flow cytometry buffer 1 % BSA and small amount (covering the tip of a spatula) NaAc
in PBS
Flow cytometry fixing solution 7 % Formaldehyde in flow cytometry buffer
For fixing cells: 1.5 ml Buffer + 1.5 ml Formaldehyde (7 %)
Immunoblot – Blocking solution 1x TBS
5 % milk powder 0.05 % Tween 20
Immunoblot – Developing solution 1 2.5 mM Luminol
0.9 mM p‐coumaric acid 10 mM Tris/HCl (pH 8.5)
Immunoblot – Developing solution 2 6 μl H2O2 (30 %)
Buffer/Solution Preparation
10 mM Tris/HCl (pH 8.5) 9 ml H2O
Immunoblot – TBS (10 x) 1.37 M NaCl
100 mM Tris/HCl (pH 7.2)
Immunoblot – Wash solution 1 x TBS
0.05 % Tween 20
Immunoblot‐ Running buffer 15.1 g Trisbase 94 g Glycine 50 ml 10% SDS
Loading buffer for DNA (10x) 6.6 g Sucrose
0.04 g Bromphenol blue dissolve in 10 ml H2O
Mini prep solution 1 50 mM Tris (pH 8.0)
10 mM EDTA (pH 8.0)
add 100 μg/ml RNase A before use
Mini prep solution 2 P2a: 400 mM NaOH
P2b: 2 % PBS mix 1:1 before use
Mini prep solution 3 3 M Kac, pH 4.8
PBS (10 x) 1.37 M NaCl
27 mM KCl 81 mM Na2HPO4
14.7 mM KH2PO4
Protein purification – Binding buffer (10 x) 0.2 M Na3PO4 pH 7,0
Protein purification – Elution buffer (10 x) 1 M Glycin‐HCl pH 2.7
Protein purification – Neutralising buffer 1 M Tris‐HCl pH 9.0
SDS PAGE – Electrophoresis buffer (5 x) 125 mM Trisbase 1.25 M Glycine 0.5 % SDS
SDS‐PAGE – Laemmli buffer (2 x) 374 mM Tris 416 mM SDS
438 mM Saccharose adjust to pH 6.8
add 1,34 mM Bromphenol blue
SDS‐PAGE – SDS separation gel (10 %) 7.9 ml H2O
6.7 ml Polyacrylamide (30 %) 5 ml Tris/SDS (pH 8.8)
200 µl APS 20 µl TEMED
SDS‐PAGE – SDS stacking gel 2.185 ml H2O
0.375 ml Polyacrylamide (30 %) 0.38 ml Tris/SDS (pH 6.8)
Buffer/Solution Preparation 30 µl APS 3 µl TEMED
SDS‐PAGE – Tris/SDS (pH 6.8) 4 x 499 mM Tris 139 mM SDS pH 6.8
SDS‐PAGE – Tris/SDS (pH 8.8) 4 x 1.5 M Tris 13.9 mM SDS pH 8.8
SE buffer 21,95 g 75 mM NaCl
5 ml 0.5 M EDTA
fill‐up to 500 ml with H2O
Silica milk (100 mg/ml) 0.1 g/ml Silica in PBS
incubation for 2 h, centrifuge, take supernatant (2x) and adjust with 3 M NaJ
TBE Agarose gel (1 %) 1 x TBE
1 % Agarose
0.5 μg/ml Ethidium bromide
TBE buffer (10 x) 1 M Tris
0.8 M boric acid 10 mM EDTA pH 8.0
TE buffer 1 ml 1 M Tris
0.2 ml 0.5 M EDTA
fill‐up to 100 ml with H2O adjust pH to 7.4
Trypan blue solution 1 x PBS
0.5 % Trypan blue
2.1.9 Media
All media except DMEM and RPMI were autoclaved and sterile antibiotics were added afterwards.
Medium Preparation
DMEM (10, 15 or 20) 1 x DMEM (High Glucose)
10, 15 or 20 % heat inactivated FCS Gold (45 min at 56 °C)
1 % Penicillin/Streptomycin
RPMI 1 x RPMI
10 % heat inactivated FCS Gold (45 min at 56 °C)
1 % Penicillin/Streptomycin
LB medium 10 mg/ml Tryptone
5 mg/ml Yeast extract 10 mg/ml NaCl
LB‐Amp Agar with IPTG and X‐Gal LB medium
15 mg/ml Agar‐Agar 0.05 mg/ml Ampicillin
Medium Preparation
0.2 % X‐Gal (2 % in Dimethylformamid) 0.024 mg/ml IPTG
LB‐Amp medium LB medium
0.05 mg/ml Ampicillin
LB‐Kan medium LB medium
0.1 mg/ml Kanamycin
LB‐Kan Agar LB medium
15 mg/ml Agar‐Agar 0.1 mg/ml Kanamycin
SOC media 20 ml/mg Tryptone
5 mg/ml Yeast extract 10 mM NaCl
2.5 KCl 10 mM MgCl2 20 mM Glucose
2.1.10 Kits
Kit Manufacturer
BigDye Terminator®v1.1 CycleSequencing Kit Applied Biosystems
pGEM‐T Easy Cloning kit Promega
QIAquick® Plasmid Midi / Maxi Kit Qiagen
ClonaCell®‐HY Hybridoma Kit Stem Cell Technologies Pierce Rapid ELISA Mouse mAb Isotyping Kit Thermo Scientific DyLight Antibody Labeling Kit (for Dylight 488, Thermo Scientific
633 and 405
2.1.11 Antibodies
Name Clone Application Manufacturer
Alexa Fluor®700 Mouse Anti‐Human CD3 SP34‐2 Flow cytometry BD V450 Mouse Anti‐Human CD4 L200 Flow cytometry BD V500 Mouse Anti‐Human CD8 RPA‐T8 Flow cytometry BD APC Mouse Anti‐Human CD11c B‐ly6 Flow cytometry BD PerCP‐Cy5.5 Mouse Anti‐Human CD14 M5E2 Flow cytometry BD APC‐Cy™ 7 Mouse Anti‐Human CD16 3G8 Flow cytometry BD PE‐Cy7 Mouse Anti‐Human CD20 L27 Flow cytometry BD FITC Mouse Anti‐Human CD56 NCAM16.2 Flow cytometry BD
PE Mouse Anti‐Human CD159a Z199 Flow cytometry Beckman Coulter FITC‐Mouse Anti‐Human γδ TCR SA6.E9 Flow cytometry Invitrogen PE‐Cy5 Goat Anti‐Mouse IgG, F(ab’)2 Flow cytometry Santa Cruz
Rabbit Anti‐Human IgG HRP Immunoblot Dako
Goat Anti‐Mouse IgG HRP Immunoblot Santa Cruz
Peroxidase Anti‐Mouse IgG ELISA Dianova
2.1.12 Oligonucleotides
Name Sequence (5’ ‐> 3’)
G23 GTTTTCCCAGTCACGAC
G24 GGATAACAATTTCACACAGG
pFUSE‐IgG1‐Fc2 fw GCGCCTACCTGAGATCACC
pFUSE‐IgG1‐Fc1 rev GGGAAGAGGAAGACTGACG
KIR pFUSE2 fw GGTGAATTCGCACACGGGTGGTCAGGAC
KIR inh pFUSE2 rev GGTGAATTCGAATGCAGGTGTCTGGGGATAC
KIR akt pFUSE2 rev GGTGAATTCACAGGCAGGTGTCTGGTGATAC
KIR bes akt pFUSE2 rev GGTGAATTCACAGGTACCTGGAGG
Name Sequence (5’ ‐> 3’)
KIR2 AcGFP fw GATGAATTCAGCACCATGTCGCTCATGG
KIR2 AcGFP rev GGTGGATCCGGATAGAAGACAACTTTCGATC
KIR9 AcGFP fw GATGAATTCAGCACCATGTCGCTCATAG
KIR9 AcGFP rev GGTGGATCCAGTCTCTTTTTGTCGG
KIR23 AcGFP rev CCAGGATCCCGCTGTTCTGTCCCCACAGG
pAcGFP fw CAAATGGGCGGTAGGCGTG
pAcGFP rev GTGGCCATTCACATCGCCAT
2.1.13 Software and Databases
Software
4 Peaks (mekentosj.com)
FlowJo 8.8.7 (Tree Star Inc.) BD FACSDiva (BD Bioscience)
Microsoft Word (Microsoft Office 2008) Microsoft Excel (Microsoft Office 2008) MEGA 5.0 (Tamura et al., 2011) ClustalX (Thompson et al., 1997)
Generation of a phylogenetic tree
Amino acid sequences were aligned with Clustal X and a phylogenetic tree was reconstructed with the neighbor joining method using JTT model, provided by the software package MEGA 5.0, with 1000 bootstrap replications.
Database
NCBI http://www.ncbi.nlm.nih.gov/
Online programme
Reverse complement http://www.bioinformatics.org/sms/rev_comp.html Translate tool http://web.expasy.org/translate/