2. Materials and methods
2.1 Materials
Name Vendor
BD Safety-Lok™ blood collection set 21G BD Biosciences, Plymouth, UK BD Vacutainer® 170 U/mL Li-Hep 10 mL BD Biosciences, Plymouth, UK BD Vacutainer® 1.8 mg/mL K2EDTA 2.5 mL BD Biosciences, Plymouth, UK BD Vacutainer® SST™ Advance K2EDTA 8.5 mL BD Biosciences, Plymouth, UK
BD Trucount® tubes BD Biosciences, San Jose, USA
Leucosep™ tubes 50 mL Greiner, Kremsmünster, Austria
Stimulation tubes round-bottom PS 12 mL Greiner, Kremsmünster, Austria FACS tubes round-bottom PS 5 mL BD Biosciences, Bedford, USA
Conical tube PP 50 mL BD Biosciences, Durham, USA
Eppendorf tubes 0.5, 1.5, 2.0 mL Safe-Lock™ Eppendorf, Hamburg, Germany Serological pipets 1, 5, 10, 25 and 50 mL BD Biosciences, Franklin Lakes, USA Pipet tips 10, 100, 1000 µL Greiner, Kremsmünster, Austria Riplate®, 96-deep-well, 1.2 mL, PP, v-bottom Ritter, Schwabmünchen, Germany
2.1.2 Reagents
Reagent Vendor/Provider Remarks
10X BD FACS™ Lysing Solution BD Biosciences, San Jose, USA 10X BD FACS™ Permeabilizing Solution 2 BD Biosciences, San Jose, USA Fixation/Permabilization Concentrate Ebioscience, San Diego, USA Fixation/Permabilization Diluent Ebioscience, San Diego, USA 10X Permeabilization Buffer Ebioscience, San Diego, USA
Beriglobin CSL Behring, King of Prussia, USA 1 mg/mL in PBS Bovine serum albumin (BSA) Roche Applied Science, Penzberg,
Germany
Brefeldin A Sigma-Aldrich, Munich, Germany 5 mg/mL in 70 % ethanol BD GolgiStop™ (Monensin) BD Biosciences, San Jose, USA
Anti-human CD28 NA/LE BD Biosciences, San Jose, USA 1 mg/mL in PBS
DMSO Sigma-Aldrich, Munich, Germany
DAPI Roche, Basel, Switzerland 1 µg/mL in PBS
Casy®ton Roche, Basel, Switzerland
EL (Erythrocyte Lysis) buffer Qiagen, Hilden, Germany
Human AB serum Lonza, Basel, Switzerland Heat-inactivated at 56 °C Live/Dead® Fixable Aqua Life Technologies, Carlsbad, USA Solved in 500 µL DMSO Live/Dead® Fixable Red Life Technologies, Carlsbad, USA Solved in 500 µL DMSO and
pre-diluted 1:100 before usage Lymphocyte separation medium LSM
1077 PAA, Pasching, Austria
Yellow fever vaccine (Stamaril®) Sanofi Pasteur MSD, Berkshire, UK Used for vaccination Yellow fever vaccine (RKI) Robert Koch-Institute, Berlin
Germany Used for stimulation
1 human dose solved with 150 µL RPMI/AB
NS4b 9mer JPT peptide technologies, Berlin,
Germany LLWNGPMAV 0.25 µg/µL in
50 % DMSO / 50 % PBS Staphylococcus enterotoxin B (SEB) Sigma-Aldrich, Munich, Germany 1.5 µg/µL in PBS Toxic shock syndrome toxin 1 (TSST1) Sigma-Aldrich, Munich, Germany 1.0 µg/µL in PBS
MATERIALS AND METHODS
CMV pp65 peptide pool JPT peptide technologies, Berlin,
Germany Swiss Prot ID.: P06725,
138 15mers, 11 aa overlap, 0.25 µg/µL per peptide solved in 50 % DMSO / 50 % PBS CMV IE-1 peptide pool JPT peptide technologies, Berlin,
Germany NCBI seq.: CAA35325.1,
120 15mers, 11 aa overlap, 0.25 µg/µL per peptide solved in 50 % DMSO / 50 % PBS EBV EBNA1 peptide pool JPT peptide technologies, Berlin,
Germany NCBI seq.: YP401677,
158 15mers, 11 aa overlap 0.25 µg/µL per peptide solved in 50 % DMSO / 50 % PBS EBV BZLF1 peptide pool JPT peptide technologies, Berlin,
Germany NCBI seq.: YP_401673,
culture medium Life Technologies, Carlsbad, USA 10X PBS pH 7.2 Life Technologies, Carlsbad, USA
EDTA Merckmillipore, Darmstadt,
Germany
BD FACSFlow™ BD Biosciences, San Jose, USA
MACSQuant Storage Solution Miltenyi, Bergisch-Gladbach, Germany
MACSQuant Washing Solution Miltenyi, Bergisch-Gladbach, Germany
MACSQuant Running Buffer Miltenyi, Bergisch-Gladbach, Germany
SPHERO™ Rainbow Calibration Particles BD Biosciences, San Jose, USA Millipore H2O
2.1.3 Kits
Kit name Vendor
Anti-Flavivirus mosaic IIFA Euroimmun, Lübeck, Germany Anti-Yellow fever virus IIFA with IgM, IgG, IgA Euroimmun, Lübeck, Germany
CMV IgG-Elisa PCS Medac, Wedel, Germany
EBV IgG-Elisa PCS Medac, Wedel, Germany
QIAamp viral RNA minikit Qiagen, Hilden, Germany
QuantiTect Virus + ROX Vial kit Qiagen, Hilden, Germany
MATERIALS AND METHODS
1X BD FACS™ Lysing Solution 10X BD FACS™ Lysing Solution 1:10 in H2O
1X BD FACS™ Permeabilizing Solution 2 10X BD FACS™ Permeabilizing Solution 2 1:10 in H2O 1X FoxP3 fixation buffer Fixation/Permabilization Concentrate diluted 1:4 with
Fixation/Permabilization Diluent
1X FoxP3 permeabilization buffer 10X Permeabilization Buffer diluted 1:10 in H2O
RPMI/AB RPMI + 10 % AB serum + 100 U/mL Penicillin + 0.1 mg/mL
Streptomycin
2.1.5 Software
Software Vendor/Provider
BD FACSDiva 6 BD Biosciences, San Jose, USA
MACSQuantify software Miltenyi, Bergisch-Gladbach, Germany Flowjo 9.7.x Tree Star, Ashland, USA
SPICE 5.3 Joshua Nozzi & Mario Roederer, Vaccine Research Center, NIAID, NIH Pestle 1.7 Mario Roederer, Vaccine Research Center, NIAID, NIH
GraphPad Prism 5.x GraphPad Software, La Jolla, USA
R 3.02 R Foundation for Statistical Computing, Vienna, Austria
2.1.6 Equipment
Device Vendor
CO2 incubator Innova CO-170 New Brunswick Scientific, Enfield, USA BD™ LSR II flow cytometer BD Biosciences, San Jose, USA
MACSQuant® analyzer Miltenyi, Bergisch-Gladbach, Germany
Eppendorf Research® pipets Eppendorf, Hamburg, Germany
Pipetboy® pipet controller Integra, Zizers, Switzerland
HERAsafe® biosafety cabinet Heraeus, Hanau, Germany
Centrifuges 5804R and 5810R Eppendorf, Hamburg, Germany
Centrifuges Microfuge® 22, Allegra® X-15R and X22 Beckman Coulter, Krefeld, Germany
Vortex Mixer VWR International, Darmstadt, Germany
CASY Model TTC cell counter Roche Applied Science, Penzberg, Germany Nalgene® Test tube rack, 16 mm for stimulation Thermo Fisher Scientific, Waltham, USA
Vacuum pump BVC 21 Vacuubrand, Wertheim, Germany
ELISA processor BEP III Siemens, Erlangen, Germany
Sysmex XE-5000 Sysmex, Kobe, Japan
MATERIALS AND METHODS
2.1.7 Optical configurations of flow cytometers
Table 2.1: Laser and optical filter settings on LSR II
LSR II
WB leukocytecounting panel Dendritic cell panel Recent thymic emigrant panel T-cell activation panel WB CD4 + T-cell stim. panel &PBMC stim. panel
Laser line Detector name Long Table 2.2: Laser and optical filter settings on MACSQuant
MACS Quant
WB leukocytecounting panel B cell panel NK cell panel
Laser line Detector name Filter Filter Filter
Blue
MATERIALS AND METHODS