BI-10 peptide synthesis procedures were identical to those mentioned in the previous section. The near infrared dye, Alexa Fluor 750 (Invitrogen), was conjugated to the N-terminus of the BI-10 peptide. This was done at room temperature in DMSO with a one-to-one molar equivalent of Alexa Fluor 750 to peptide in the presence of 10 equivalent of triethylamine (Et3N). The conjugating reaction was monitored by analytical HPLC and was completed in 24 hours. The crude Alexa Fluor 750-peptide conjugate (BI-10AF750) was purified by C18-RP HPLC (Vydac 218-TP510). The purity of final product was checked by analytical HPLC to be in excess of 95%, and confirmation of the molecular mass was determined by MALDI mass spectroscopy (MNa+ 1946.8Da, found 1947.1Da).
Alexa fluor 750
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Alexa Fluor 750 is a fluorescent dye that can be used for labeling proteins, nucleic acids, and other biomolecules. It has an excitation maximum at 749 nm and an emission maximum at 775 nm, making it suitable for detection in the near-infrared region of the spectrum.
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Lab products found in correlation
Alexa fluor 700, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
27 gauge butterfly catheter, by Abbott (2 mentions)
Bs 8000, by Braintree Scientific (2 mentions)
Lb981 nightowl, by Berthold Technologies (2 mentions)
Multi immersion oil water objectives, by Olympus (2 mentions)
Microtome, by Leica (2 mentions)
Citrate buffer ph 6, by Merck Group (2 mentions)
Ra3 6b2, by Abcam (2 mentions)
Zen software, by Zeiss (2 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (2 mentions)
Image studio lite software, by LI COR (2 mentions)
Complete mini, by Roche (2 mentions)
Ivis lumina 3, by PerkinElmer (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Aldefluor, by STEMCELL (1 mentions)
Live dead fixable dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cd140b, by BioLegend (1 mentions)
17 dimethylaminoethylamino 17 demethoxygeldanamycin 17 dmag, by InvivoGen (1 mentions)
40 protocols using alexa fluor 750
1
Alexa Fluor 750 Labeling of BI-10 Peptide
2
Characterization of Mammary Cell Subpopulations
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Mammary cells were stained for CD44 and CD24 expression and ALDH activity as described previously (Colacino et al., 2016 (link)). In brief, single mammary cells were first incubated with a lineage-depletion cocktail that consisted of biotinylated antibodies targeted against CD45, HLA-DR, CD14, CD31, CD41, CD19, CD235a, CD56, CD3, CD16, and CD140b (all from eBioscience, except for CD140b [Biolegend] and CD41 [Acris]). Next, cells were stained with Alexa Fluor 750-tagged streptavidin, LIVE/DEAD Fixable Dead Cell Stain (Invitrogen), CD24 (Biolegend), CD44 (Becton Dickinson), and Aldefluor (STEMCELL Technology). Single-color and isotype controls were included for compensation and gating purposes. Aldefluor-positive gating was based on DEAB (negative) controls. Flow-cytometry data analysis was performed with FlowJo software version 10.0.8.
3
HER2-Targeted Theranostic Affibody Probe
4
In Vivo Molecular Imaging of Fluorescent Probes
5
Cloning and Labeling of Anti-DR5 Antibody
6
In Vivo Molecular Imaging of Fluorescent Probes
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Prior to imaging, each mouse was anesthetized using a mixture of acepromazine (0.1 mg/kg, i.m.) and torbugesic (0.1 mg/kg, i.m.). A 27½ gauge butterfly catheter (Abbott Laboratories) flushed with heparin was inserted in the tail vein and secured. The catheter was connected to a syringe filled with BI-10AF750 peptide in PBS, which was mounted on a programmable infusion pump (BS-8000; Braintree Scientific, Inc.). The mouse and infusion pump was positioned within the light-tight box of the optical imager (LB981 NightOWL, Berthold) used for the measurement of the NIR emitting molecular Alexa Fluor 750 (Invitrogen). The excitation source was filtered using a HQ710/75X bandpass filter (Chroma Technologies Corp.) and uniformly illuminated the field of view of the mouse. The emission spectrum was filtered using a HQ810/90M bandpass filter to enhance the Alexa Fluoro 750 fluorescence relative to the auto fluorescence signal. Optical images were acquired prior to injection, every 3 seconds during the injection of the probe (50 μL/min), and every 60 seconds over the next 60–90 minutes using a 10 ms exposure time. Additional images were taken 4, 8, 12, 24 and 48 hours post-injection for the mice in the first group, while images of the mice in the second group were acquired at 6 hours post-injection using a 40 msec exposure time.
7
Intravital Microscopy of Foreign Body Reaction
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For intravital microscopy, mice were anesthetized with isofluorane and stably mounted onto a temperature-controlled platform (37°C). FBR elicited by an implanted mPCL-CaP scaffold was monitored using a custom intravital multiphoton microscope (LaVision BioTech)31 (link) with three Ti:Sapphire lasers (Chameleon-XR, Coherent) and two Optical Parametric Oscillators (APE/Coherent), resulting in a tuneable excitation range from 800 to 1300 nm (Fig S1 ). Multi-spectral detection was performed using up to 5 backward or 2 forward photomultipliers (PMTs) using up to three excitation wavelengths in two consecutive scans, to separate the following excitation and emission channels: GFP (920 nm; 525/50 nm); Hoechst 33342 (920 nm; 450/60nm), Rhodamine (1090 nm; 595/40 nm), SHG (1090 nm; 525/50 nm), THG (1180 nm; 387/15 nm) and AlexaFluor750 (1180 nm; 810/90 nm).
For intravital detection, long-working distance 16x NA 0.8 water or 25x NA 1.05 multi-immersion oil/water objectives (Olympus) were used. Sequential 3D stacks were obtained with 5–10 μm step-size reaching up to 200 μm penetration depth. Images were acquired in a random fashion within the subcutaneous tissue up to the dermis. Perfused blood vessels were visualized by i.v. injection of Rhodamine- or AlexaFluor750-conjugated dextran (70 kD; Invitrogen; 1 mg/mouse). mPCL-CaP scaffolds, in vitro, were analyzed using SHG and THG imaging.
For intravital detection, long-working distance 16x NA 0.8 water or 25x NA 1.05 multi-immersion oil/water objectives (Olympus) were used. Sequential 3D stacks were obtained with 5–10 μm step-size reaching up to 200 μm penetration depth. Images were acquired in a random fashion within the subcutaneous tissue up to the dermis. Perfused blood vessels were visualized by i.v. injection of Rhodamine- or AlexaFluor750-conjugated dextran (70 kD; Invitrogen; 1 mg/mouse). mPCL-CaP scaffolds, in vitro, were analyzed using SHG and THG imaging.
8
Optoacoustic imaging of fluorescent dye
9
In vivo and ex vivo Near-Infrared Fluorescence Imaging
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In vivo and ex vivo near-infrared fluorescence imaging was performed using the Pearl Impulse imaging system (LI-COR, Lincoln, NE). NOD mice were anesthetized with ketamine/xylazine at 50 and 5 mg/kg body weight, respectively. In vivo imaging was performed at 6, 24, and 48 hours after intraperitoneal (i.p.) administration of 3.5 mg of fluorescence labeled PS-liposomes (Alexa Fluor 750, Invitrogen) in 200 μl of saline solution. At the end of every checkpoint, perigonadal adipose tissue, kidney, spleen, pancreas, pancreatic lymph nodes, mesenteric lymph nodes, liver, mediastinal lymph nodes and thymus were harvested, washed in PBS, and imaged ex vivo using the Pearl Impulse system (LI-COR). Fluorescent signal intensity was semi-quantitatively assessed: Fluorescence level was normalized by subtracting the background and represented as a relative index of fluorescence in each organ (RFU) / grams of tissue.
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In vivo and ex vivo near-infrared fluorescence imaging was performed using the Pearl Impulse imaging system (LI-COR, Lincoln, NE). NOD mice were anesthetized with ketamine/xylazine at 50 and 5 mg/kg body weight, respectively. In vivo imaging was performed at 6, 24, and 48 hours after intraperitoneal (i.p.) administration of 3.5 mg of fluorescence labeled PS-liposomes (Alexa Fluor 750, Invitrogen) in 200 μl of saline solution. At the end of every checkpoint, perigonadal adipose tissue, kidney, spleen, pancreas, pancreatic lymph nodes, mesenteric lymph nodes, liver, mediastinal lymph nodes and thymus were harvested, washed in PBS, and imaged ex vivo using the Pearl Impulse system (LI-COR). Fluorescent signal intensity was semi-quantitatively assessed: Fluorescence level was normalized by subtracting the background and represented as a relative index of fluorescence in each organ (RFU) / grams of tissue.
10
Multi-parameter flow cytometry profiling
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