Alexa 750 dyes

These assays were performed at the Human Immune Monitoring Center at Stanford University. Briefly, PBMCs were suspended at 0.1×10⁶ viable cells and stimulated with T cell receptor (TCR) stimuli (CD3/CD28 Dynabeads) or B cell receptor (BCR) stimuli (anti-human IgG, anti-human IgM and H₂O₂) and incubated at 37°C for either 30 minutes (TCR) or 4 minutes (BCR). The PBMCs were then fixed with paraformaldeyde, and permeabilized with methanol. Cells were bar-coded using a combination of Pacific Orange and Alexa-750 dyes (Invitrogen, Carlsbad, CA) and then stained with the following antibodies (all from BD Biosciences, San Jose, CA): CD3 Pacific Blue, CD4 PerCP-Cy5.5, CD20 PerCp-Cy5.5, CD33 PE-Cy7, CD45RA Pacific Orange, p38 FITC, pPLCγ2 PE, pSTAT-5 PE-TX-Red, and pERK1/2- APC. Cells were collected (100,000/sample) using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v9.3 by gating on live cells based on forward versus side scatter profiles, then on singlets using forward scatter area versus height, followed by cell subset-specific gating of the 90^th percentile. Fold change was calculated over basal phospho-protein levels to assess differences following BCR and TCR stimulation.

This assay was performed by the HIMC at Stanford University. PBMCs were thawed in warm medium, washed twice and resuspended at 0.5 × 10⁶ viable cells ml⁻¹. Then, 200 μl of cells were plated per well in 96-well deep-well plates. After resting for 1 h at 37 °C, cells were stimulated by adding 50 μl of cytokine (IFN-α, IL-6, IL-10 or IL-2) and incubated at 37 °C for 15 min. PBMCs were then fixed with paraformaldehyde (PFA), permeabilized with methanol and stored at −80 °C overnight. Each well was barcoded using a combination of Pacific Orange and Alexa-750 dyes (Invitrogen) and pooled in tubes. Cells were washed with FACS buffer (PBS supplemented with 2% FBS and 0.1% sodium azide) and stained with the following antibodies (all from BD Biosciences): CD3 Pacific blue, CD4 PerCP-Cy5.5, CD20 PerCp-Cy5.5, CD33 PE-Cy7, CD45RA Qdot 605, pSTAT-1 AlexaFluor488, pSTAT-3 AlexaFluor647 and pSTAT-5 PE. The samples were then washed and resuspended in FACS buffer. Then, 100,000 cells per stimulation condition were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v.9.3 by gating on live cells based on forward versus side-scatter profiles, then on singlets using forward scatter area versus height, followed by cell subset-specific gating.