Synthesis of the aptamers and conjugation to dyes and drugs was performed as described previously [12 (link)]. Briefly, the E3 (GGC UUU CGG GCU UUC GGC AAC AUC AGC CCC UCA GCC) and C36 (GGC GUA GUG AUU AUG AAU CGU GUG CUA AUA CAC GCC) aptamers were synthesized using 2′-F-modified pyrimidines and 2′OH purines on an inverted dT CPG column by solid-phase synthesis. A 5′thiol C6 disulfide linker was added to the aptamer and reduced by incubating with 500 nM TCEP. The maleimide activated dyes, AF488, DL650, or AF750 (Invitrogen, Carlsbad, CA) or the auristatin derivates maleimide-caproyl-valine-citrulline-p-aminobenzylcarbamate-MMAE or maleimidocaproyl-MMAF (Levena Biopharma, San Diego, CA, USA) were added to the free thiol end of the aptamer. Conjugation efficiency was confirmed via analytical HPLC.
Af750
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AF750 is a compact and versatile lab equipment designed for a range of applications. It features a durable construction and precise temperature control capabilities. The core function of the AF750 is to provide a reliable and consistent environment for various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pearl impulse imaging system, by LI COR (1 mentions)
Amicon ultra 4, by Merck Group (1 mentions)
Du 640, by Beckman Coulter (1 mentions)
Anti cd19 pe cy7, by BD (1 mentions)
Facscelesta, by BD (1 mentions)
Anti cd4 percp, by BD (1 mentions)
Anti cd3 bv650, by BD (1 mentions)
Anti igm pe, by BD (1 mentions)
Anti cd38 fitc, by BD (1 mentions)
Version 10, by FlowJo (1 mentions)
5 protocols using af750
1
Aptamer Synthesis and Conjugation
2
Multi-Modal Tissue Imaging Protocol
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For IF staining, tissue was prepared using the same protocal in IMC staining, then stained overnight at 4 °C with a single metal-conjugated primary antibody (CD3, CD4, CD169, or CD61 in Supplemental Table 5) . The single-stained tissue was washed, then stained with secondary antibody (donkey anti-rabbit AF647 or goat anti-mouse AF750, Invitrogen, 2 mg/mL diluted 1:400 in 0.5% BSA in PBS) at room temperature 1 h, followed by additional washing in PBS and DAPI (1 μg/mL) staining. Slides were mounted with SlowFade Glass antifade reagent (ThermoFisher) and # 1 1/2 coverslips. Images were acquired using Leica DMi8 inverted widefield microscope with Lumencor SOLA SE U-nIR light engine, DAPI/FITC/TRITC/Cy5/Cy7 filters, DFC9000 GT sCMOS camera, PL APO 20x/0.80 objective and LAS X software (version 3.7.3.23245). After image acquisition, coverslips were removed with gentle agitation in PBS, then Ir-intercalator staining, washing and drying performed as above for subsequent Hyperion data acquisition.
3
In Vivo and Ex Vivo Imaging of Liraglutide
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Liraglutide was conjugated to AlexaFluor750 (AF750, Invitrogen) using a standard method (Thiol-Reactive Probes, Invitrogen). In vivo and ex vivo near-infrared fluorescence imaging was performed (Pearl Impulse imaging system, LI-COR) in NOD mice anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) at 15 and 60 min after the s.c. administration of 1 mg/kg of AF750-liraglutide in 50 μl of PBS. At the end of each checkpoint, spleen, stomach, fat, heart, liver, pancreas, lungs, kidney, salivary glands, thymus, and bladder were imaged ex vivo. Fluorescent signal intensity was semi-quantitatively assessed: the levels were normalized by subtracting the background and represented as a relative index of fluorescence in each organ per gram of tissue.
4
Fluorescent Labeling of Trastuzumab
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Humanized monoclonal antibody against HER2 trastuzumab was obtained from Genentech and conjugated to AF700 or AF750 (Thermo Fisher Scientific) through monoreactive N-hydroxysuccinimide ester to lysine residues in the presence of 100 mM Na bicarbonate, pH 8.3, according to manufacturer’s instructions. The probes were purified by using Amicon Ultra-4 centrifugal filter units (Sigma Z648035, MWCO 30 kDa). After extensive washes with PBS, the probes are reconstituted in PBS, protein concentration is measured and normalized to 1 mg/mL. The degree of labeling of the probes was assessed by spectrophotometer DU 640 (Beckman Coulter, Fullerton, CA, USA). The average degree of labeling was no more than 2 fluorophores per molecule. All probes were filter sterilized and stored at 4 °C without preservative.
5
Flow Cytometry Analysis of PBMCs
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All FACS measurements were conducted using a FACS Celesta (BD Biosciences, Franklin Lakes, NJ, USA). PBMC were thawed rapidly at 37°C. After washing with cold PBS, cells were counted using a Neubauer counting chamber and diluted to desired concentrations. For FACS analysis, PBMC were blocked with human serum for 30 min at 4°C and incubated with a pre-titrated antibody cocktail including RTX-AF647 as well as AF750 (Thermo Fisher Scientific), for live-dead staining. For FACS analysis, in each sample at least 200,000 cells were regularly acquired. If not otherwise indicated, all antibodies were obtained from BioLegend (San Diego, CA, USA). The following antibodies and fluorophores were used: V450 anti-CD27 (clone: M-T271), V500 anti-IgD (clone: IA6-2), BV650 anti-CD3 (clone: OKT3), BV785 anti-CD45 (clone: HI30), FITC anti-CD38 (clone: HIT2; BD BioSciences), PE anti-IgM (clone: MHM-88), PerCP anti-CD4 (clone: L200), PE-Cy7 anti-CD19 (clone: HIB19), AF700 anti-CD8a (clone: HIT8a). PBMC were washed twice and measured using a FACS Celesta (BD BioSciences). Graphical analysis was performed using FlowJo version 10.6.1 (FlowJo, Ashland, OR, USA). Of note, in contour plots not all cells are depicted as single dots. For gating strategy see supplemental information (Figure S1
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