Sulfo cy3 nhs ester

192 oligonucleotides that are complementary to the 8-nt cell barcodes (Supplementary Table S2) with 3′-amino modifications were synthesized and purified (Sigma-Aldrich), then resuspended in water at 200 μM. To generate probe mixtures corresponding to each bit in the binary code, oligonucleotides labeled with ‘1’ were taken (Fig. 1D, Supplementary Table S3), pooled and resuspended in 0.1 M sodium tetraborate (pH 8.5) coupling buffer at a final concentration of 22 μM with 0.6 μg/μL reactive fluorophore. Sulfo-CY5 NHS ester (Lumiprobe, cat# 21320) was coupled with S oligo pools, and Sulfo-CY3 NHS ester (Lumiprobe, cat# 23320) was coupled with Q oligo pools overnight at room temperature. Excess fluorophores were removed and oligos were recovered by ethanol precipitation (80% Ethanol, 0.06 M NaCl, 6 μg/mL glycogen). The concentration of probes was quantified using a NanoDrop (Thermo Scientific). Probe pools were diluted such that each probe had a final concentration of ~ 20 nM, and the two, distinctly labeled probe pools were mixed together for each binary code bit prior to use.

The monoclonal antibody CR3022 was purchased from Absolute Antibody (#Ab01680-10.0) as an IgG1. F(ab’)2 was produced using the Genovis FragIT kit (#A2-FR2-100, Genovis) according to manufacturer’s protocol. Briefly, 1-5 mg of IgG1 was added to the FragIT column after equilibration and allowed to rock at room temperature for 45 minutes, the column was then centrifuged to elute IgG fragments. Fc fragments were removed by passing through a CaptureSelect Fc Column. F(ab’)2 fragments were eluted and collected. F(ab’)2 production was checked through SDS-PAGE. The resulting gel was first imaged under white light to show the fluorophore labeling of the F(ab’)2 before staining with SimplyBlue SafeStain (Thermo-Fisher, #LC6060). For labeling, 3 mgs of purified CR3022-F(ab’)2 was combined with 60ug of sulfo-Cy5 NHS ester (#53320, Lumiprobe), sulfo-Cy3 NHS ester (#51320, Lumiprobe), or AF647-NHS ester (#A20106, Thermo-Fisher) in PBS with 100mM sodium bicarbonate and gently rocked at room temperature for 1 hour as previously described (43 (link)). Solutions were then passed through a Zeba column (Thermo-Fisher, #89882) to remove free dye. Labeled F(ab’)2 was filtered through a 0.22-μm filter and stored at 4°C in the dark. Labeled F(ab’)2 to be infused into macaques was tested using the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo-Fisher, #88282).