Fig. 2
Validation of a 48-well single-cell chemical biology assay for DNA damage response mechanism class. Design of a checkerboard validation experiment using Kasumi cells and a DNA-active natural product. a Overview of 48-well fluorescent cell barcoding and debarcoding validation. Compounds and vehicle are added in a checkerboard pattern to 48 wells, and cells are added and incubated prior to being barcoded using dye gradients of N-hydroxysuccinimide functional Pacific Orange (PO) and Pacific Blue (PB). Cells are stained with Ax700, fixed, permeabilized, and pooled prior to immunoassay with antibodies tagged with nonoverlapping fluorescent dyes. Cells are analyzed by flow cytometry and gated selecting (i) intact, single cells, (ii) PO to reveal columns, and (iii) PB to reveal rows and generate populations for each well. PO* and PB* denote dye intensity after correcting for cell morphology using a protocol from ref. 67. A data set using Kasumi cells for an etoposide checkerboard assayed with γH2AX binding, which is a marker for DNA damage-associated activity is shown here. b Graphical representation of well coordinates generated from pooled cells by gating on forward scatter and γH2AX and then debarcoding as above to reveal the checkerboard pattern. c Biaxial plots of individual wells representing a condition, as in Fig. 1a. d Percent γH2AX positive for each well was calculated using a cutoff of 103.2 to determine statistical effect size (Z′). A similar demonstration with etoposide vs. cCasp3 is shown in Supplementary Fig. 1
