Maxpar antibody conjugation kit

CyTOF allows multidimensional relative protein quantification for single-cell datasets and we adapted it for platelets using a customized mass cytometry panel of 21 antibodies (Table 1). For custom-made antibody conjugations, 100 mg of carrier-free antibody was coupled to metal-labeled X8 polymer according to the manufacturer’s instructions (Fluidigm). Briefly, using the MaxPAR antibody conjugation kit (Fluidigm) following the manufacturer’s recommended protocol, six antibodies were conjugated to isotopically enriched lanthanide metals. After labeling, the antibodies were stored in an antibody stabilization buffer (Boca Scientific, Westwood, MA, USA) at 4 °C. The other antibodies were pre-conjugated, CyTOF-ready, and commercially available (Fluidigm Sciences). Please see the Supplemental Materials for the reagent list. All custom-conjugated antibodies were validated with calibration beads. In detail, 0.5 µl of the conjugated antibody was added to one drop of beads and incubated for 15 min. After two washing steps with 1.5 ml PBS at 300 × g for 10 min, the mixture was washed twice with de-ionized water at 300 × g for 10 min, and resuspended in 200 µl water until acquisition.

Purified antibodies for mass cytometry were obtained in carrier/protein-free buffer and then coupled to lanthanide metals using the MaxPar antibody conjugation kit (Fluidigm Inc.) as per the manufacturer’s recommendations. Following the protein concentration determination by measurement of absorbance at 280 nm on a nanodrop, the metal-labeled antibodies were diluted in Candor PBS Antibody Stabilization solution (Candor Bioscience, Germany) for long-term storage at 4°C. Antibodies used are listed in Supplementary Table S1.

The mass cytometry antibody panel included 28 antibodies that were used for phenotyping of immune cell subsets and 11 antibodies for the functional characterization of immune cell responses (Table S2). Antibodies were either obtained preconjugated (Fluidigm, Inc.) or were purchased as purified, carrierfree (no BSA, gelatin) versions, which were then conjugated in-house with trivalent metal isotopes utilizing the MaxPAR antibody conjugation kit (Fluidigm, Inc.). After incubation with Fc block (Biolegend), pooled barcoded cells were stained with surface antibodies, then permeabilized with methanol and stained with intracellular antibodies. All antibodies used in the analysis were titrated and validated on samples that were processed identically to the samples used in the study. Barcoded and antibody-stained cells were analyzed on the mass cytometer.