V. Methods
2.2 Protocols for cell staining and flow cytometry analysis
20,000-‐150,000 cells per well/per staining were transferred in a 96-‐well round-‐bottom plate and centrifuged (805 x g for 1min) to pellet the cells. The supernatant was discarded and the cells were washed once with 200 µL flow wash buffer (PBS containing 5 % FCS and 0.02 % NaN3). The single stain antibodies or antibody staining mixes were prepared on ice and anti-‐
bodies were diluted in flow wash buffer. The staining was carried out in a total volume of 20 µL per well at 4 °C in the dark for 30min. Then, the cells were washed once with flow wash buffer and the cell pellet was resuspended in 200-‐250 µL flow wash buffer. 10 µL propidium
iodide was added per staining as live/dead indicator, immediately before recording the samples on a BD LSRFortessa flow cytometer in combination with the software BD FACSDIVA 7.0 and analysis with FlowJo Software. If possible, 10,000-‐20,000 events were recorded per sample and propidium iodide positive cells were excluded from the analysis. Cells were measured in single-‐tubes, except for the flow cytometry-‐based cell surface screening (sec-‐
tion 2.2.2) and the testing of hybridoma supernatants (section 3.1.4), for these experiments the HTS Plate Loader unit of the BD LSRFortessa was used for acquisition.
If Aqua staining was used as the live/dead indicator, the cells were washed twice with ice-‐
cold PBS and then stained with 100 µL Aqua (1:1000 in ice-‐cold PBS) at 4 °C in the dark for 30min, before the staining with the respective antibodies. Aqua positive cells were then ex-‐
cluded from the analysis.
If cells needed to be fixed, they were washed once with flow wash buffer after the staining.
Then the cells were incubated with 100 µL Fix-‐Solution (BD Cytofix/Cytoperm Kit) at 4 °C in the dark for 20min. The cells were washed twice with 200 µL of flow wash buffer and kept at 4 °C in the dark until the measurement.
If a multicolor staining was applied to the sample, a compensation experiment was set up in advance. Therefore, per antibody, a mixture of 100 µL flow wash buffer with one drop of BD Comp Beads (Anti-‐Mouse Ig, κ; Anti-‐Rat Ig, κ; depending on the subtype of the antibody), one drop of Negative control beads and 2-‐3 µL antibody was prepared and incubated at RT in the dark for 30min. 2 mL flow wash buffer was added and bead-‐antibody mixtures were centrifuged at 200 x g for 10min. The bead pellets were then resuspended in 200 µL fresh flow wash buffer each and compensation beads were recorded in the compensation setup. If Aqua was used, one drop of ArC Amine reactive beads was allowed to adjust to RT for 5min in a tube. 2 µL of Aqua was added, the mixture was vortexed and incubated at RT in the dark for 30min. One drop of Negative Control Beads and 2 mL of flow wash buffer was added and then centrifuged at 200 x g for 10min. The bead pellet was resuspended in 200 µL of fresh flow wash buffer. The final compensation was then calculated with FACSDIVA 7.0 software and could be applied for multicolor experiments.
2.2.1 Detailed settings for individual experiments using flow cytometry
Staining of PBMCs was performed on a cell number of 150,000 cells/staining. If isolated T cells were stained for flow cytometry, the number of cells was adjusted to 100,000 cells/staining. Except the flow cytometry-‐based cell surface screening (see 2.2.2), consisting of only one conjugated antibody per staining and due to the limitation of the total available cell number of one blood donor, the number of cells was adjusted to 20,000 cells/staining.
Purity and viability of naive T cells after the isolation process was always checked via CD45RA+/CD45RO-‐ staining of the resulting cell population. During the activation of the naive CD4+ T cells via anti-‐CD3/anti-‐CD28 stimulation the activation marker CD69 was moni-‐
tored to ensure proper activation. The efficiency of oxidation/biotinylation reaction (PAL-‐
qLC-‐MS/MS) (section 2.1.1) was measured via Streptavidin-‐PE antibody staining. Detailed information for the respective antibodies is listed in Table M2.
2.2.2 Flow cytometry-‐based cell surface screening and data analysis
This experiment was performed in cooperation with the Institute of Virology, Helmholtz Cen-‐
ter Munich (Prof. Dr. Michael Schindler and Dr. Herwig Koppensteiner). A targeted cell sur-‐
face antigen screening, based on monoclonal antibodies and flow cytometry, was performed with the LEGENDScreen Human Cell Screening (PE) Kit. This kit includes 96 well plates, which are pre-‐coated with one lyophilized monoclonal PE-‐conjugated antibody per well. It contains 332 antibodies against human cell surface antigens and 10 Ig isotype controls (mouse, rat, hamster). Naive CD4+ T cells from 3 different human donors (n=3, D5-‐D7) were taken in their naive form and in addition cells were activated with anti-‐CD3/anti-‐CD28 for 3 and 24h for this experimental setting, generating 9 samples (cells of 3 donors, 3 time points each) for analysis in total. The cells were washed with cold PBS and centrifuged at 290 x g for 10min directly after isolation, respectively after the end of the stimulation. The cells were resus-‐
pended in Cell Staining Buffer, included in the kit, and kept on ice. The antibody-‐coated 96-‐
well plates were centrifuged at 805 x g for 5min, followed by dissolving the antibodies in 75 µL of ddH2O. The plates were then incubated at RT in the dark for 15min. After incuba-‐
tion, two times 25 µL were transferred from the original 96-‐well plate to new 96-‐well plates.
20,000 cells in 75µL volume were then transferred to each well containing 25 µl of dissolved antibody, resuspended and incubated at 4°C in the dark for 30min. The cells were washed once with 200 µL cell staining buffer per well and then resuspended in 100 µL fixation buffer, provided in the kit. The cells were incubated at RT in the dark for 15min and then washed again with 200 µL cell staining buffer. The fixed cells were resuspended in 200 µL cell stain-‐
ing buffer and kept at 4 °C in the dark until data acquisition.
The resulting data was analyzed using BD FACSDIVA 7.0 and FlowJo Software. Antibody sig-‐
nals were considered as positive when the MFI was higher than the highest measured Ig iso-‐
type control. In addition, it was necessary to obtain a positive signal on the cells of at least two donors (otherwise stated in Table 2), if the protein was considered for the cell surface protein atlas.
2.3 Analysis of proteomic results: unsupervised clustering by GProx and Gene ontology