Fig. 2: Validation and quantification of ACE for mass cytometry signal amplification.

a, HEK293T cells transiently transfected with a GFP-encoding plasmid were used to validate the ACE amplification method. b, Signal amplification by ACE (y axis) through 1–500 thermal cycles was compared with counterstaining using a secondary antibody conjugated with the conventional protocol (x axis) to validate the specificity and quantify the amplification efficiency of ACE. Pearson correlation coefficients between ACE and the regular secondary antibody signals were calculated for each condition as a measure of ACE specificity. c, Data were divided into ten equal-width bins according to their GFP expression levels shown by the secondary antibody. Bins 1–3 reflect the untransfected cells as internal controls. Bin 10 shows the cells with the highest GFP expression levels. d, Bin medians across 1–500 thermal cycles. e, Ratio of each bin median over the median in bin 10 across 1–500 thermal cycles. f, GFP ion counts generated with ACE after one or two rounds of branching were compared with that from a linear amplification without branching in a ERK2–GFP transient expression experiment using ultralow amount of the GFP antibody (10 ng ml⁻¹). A conventionally conjugated anti-ERK2 antibody was used as counterstaining. g, To examine the orthogonality of ACE, GFP-expressing HEK293T cells were stained individually with anti-GFP ACE antibodies conjugated to 33 initiator sequences before they were barcoded, pooled and processed through the ACE protocol in the same tube, and then analyzed on a mass cytometer. h, Data were then debarcoded to allow pairwise analysis of potential ACE crosstalk. Ion counts generated by a detector in all conditions (columns) were normalized to 0–1 before the ratio between any unmatched initiator–detector pair (for example, Ab-initiator 1–Detector 2*-¹⁴²Nd) and the true signal (for example, Ab-initiator 1–Detector 1*-¹⁴¹Pr) was calculated. On average, ACE has 1.02% of crosstalk signal. Four pairs of probes were detected with crosstalk degrees over 10% (Initiator 2–Detector 3*, Initiator 4–Detector 5*, Initiator 7–Detector 8*, Initiator 20–Detector 21*). 1°, primary; 2°, secondary; Ab, antibody.