Sulfo cy5 nhs ester

Histones from calf thymus, bovine serum albumin, N-isopropylacrylamide (NIPAm), sodium dodecyl sulfate (SDS), fluoresceinamine isomer, and poly(ethylene glycol) methyl ether methacrylate (average M_n 500, 1500, and 4000) were purchased from Sigma Aldrich. N,N’-methylenebisacrylamide (BIS) was from Fluka; N-t-butylacrylamide (TBAm) was from ACROS ORGANICS. [2,3-14C]-Acrylamide was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Histone H1, H2A, H2B, H3.3 and H4 (Human, Recombinant) were purchased from New England Biolabs, Inc., (MA, USA). Sulfo-Cy5 NHS ester was purchased from Lumiprobe (FL, USA). 5-(and 6)-carboxytetramethylrhodamine, succinimidyl ester (NHS-Rhodamine) was purchased from Thermo Fisher Scientific, Inc.

Synthesized WT or mutant ITGα5-cyto peptide was mixed with Sulfo-Cy5 NHS ester (Lumiprobe) at 1:1 molar ratio in reaction buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl₂) and incubated for 2 h at 25 °C. For the control, Tris (pH 8.0) was mixed with Sulfo-Cy5 NHS ester at 7:1 molar ratio in reaction buffer, as ITGα5-cyto-WT peptide has 7 -NH₂ groups. To fully quench the reaction, 20-fold Tris (pH 8.0) was added to react with the excess free dye for 20 min and then the mixture was centrifuged at 13,000 rpm for 10 min to remove the precipitate. The supernatant was used for the following dcFCCS assay.

Bacteriophage Qβ (20 mg) was labeled with Cy5 through conjugation via a 500-fold molar excess of sulfo-Cy5 NHS ester (Lumiprobe) in 0.5 mL 0.1 M potassium phosphate buffer (referred to as KP buffer; K₂HPO₄ and KH₂PO_4, pH 8.3), for 4 h at room temperature. The unreacted components were removed by centrifugal filtration as above, and the Cy5-Qβ particles were dialyzed against and then stored in deionized water.

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).