The NIRF probe targeting CD68+ macrophages was generated by coupling an amine-reactive NIR fluorochrome (NHS ester), VivoTag 680 (excitation peak 665 ± 5 nm, emission peak 688 ± 5 nm) (Perkin-Elmer), to a rat anti-mouse CD68 antibody (AbDSerotec) according to manufacturer’s instructions. In brief, VivoTag 680 was dissolved in dry dimethyl sulfoxide (Sigma-Aldrich) at a concentration of 10 mg/ml. Prior to the labeling, the buffer of the antibody was exchanged by dialysis into conjugation buffer (50 mM carbonate/bicarbonate buffer, pH 8.5) using Slide-A-Lyzer dialysis cassettes (AbD Serotec) according to the protocol provided by the manufacturer. After buffer exchange, 30 μl of VivoTag 680 was added to the rat anti-mouse CD68 antibody. Following 1 h of incubation at room temperature protected from the light, the NIRF CD68 probe (approximately 151 kDa) was separated from free fluorescent dye (approximately 1 kDa) and antibody oligomers (larger than 300 kDa) by fast protein liquid chromatography using a Superdex 200 resin in a pre-packed 10/300 GL column (GE Healthcare). Probe concentration was determined using a BCA Protein Assay Kit (Uptima) according to the protocol provided by the manufacturer.
Dry dimethyl sulfoxide
Manufactured by Merck Group
Sourced in United States
Dry dimethyl sulfoxide is a laboratory chemical used as a solvent in various scientific and industrial applications. It is a colorless, odorless, and aprotic polar solvent with a high boiling point. The product is designed to meet the purity and quality standards required for use in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Vivotag 680, by PerkinElmer (1 mentions)
Superdex 200 resin, by GE Healthcare (1 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Aβ1 42, by Eurogentec (1 mentions)
Aβ1 42, by Bachem (1 mentions)
Melamine, by Merck Group (1 mentions)
Imidazole, by Merck Group (1 mentions)
Hexadecyltrimethylammonium bromide, by Merck Group (1 mentions)
Anhydrous calcium chloride, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Toluene, by Thermo Fisher Scientific (1 mentions)
Tetraethyl orthosilicate, by Merck Group (1 mentions)
4 dimethylaminopyridine, by Thermo Fisher Scientific (1 mentions)
Glycidyl methacrylate, by Tokyo Chemical Industry (1 mentions)
3 trimethoxysilyl propylacrylate, by Tokyo Chemical Industry (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Bisphenol a diglycidyl ether, by Merck Group (1 mentions)
7 protocols using dry dimethyl sulfoxide
1
NIRF Macrophage Targeting Probe Synthesis
2
Oligomeric Amyloid-Beta Preparation
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Oligomeric amyloid β (Aβ1-42) was prepared as reported previously [24 (link)]. Briefly, Aβ1-42 (ABX, Radeberg, Germany) was initially dissolved in hexafluoroisopropanol (HFIP, Sigma, St. Louis, MO, USA) to a concentration of 1 mM. For the aggregation protocol, the peptide was resuspended in dry dimethylsulfoxide (5 mM; Sigma, St. Louis, MO, USA). Hams F-12 (PromoCell, LabClinics, Barcelona, Spain) was added to adjust the final peptide concentration to 100 μM to obtain oligomers (4°C for 24 h). Monomeric Aβ was dissolved in PBS to a concentration of 100 μM.
3
Oligomeric Amyloid-β Preparation
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Aβ1–42 was purchased from Eurogentec (AnaSpec, Fremont, CA, USA; AS-64129-1), and oligomeric amyloid was prepared as described in Dahlgren et al. [31 (link)]. Briefly, Aβ lyophilized with a hexafluoroisopropanol film was dissolved in dry dimethyl sulfoxide (Sigma-Aldrich Co., Seoul, Korea) to obtain a concentration of 1 mM and sonicated for 180 s. Ham’s F-12 (Invitrogen, Carlsbad, CA, USA) was then added to a final concentration of peptide 0.1 mM, and the samples were rotated on a rotary shaker at 4 °C for 7 days.
4
Amyloid-β Peptide Aggregation Protocols
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Oligomeric and fibrillar amyloid-β (Aβ1–42) was prepared as reported previously19 (link),56 (link). Briefly, Aβ1–42 (Bachem, Germany) was initially dissolved in hexafluoroisopropanol (HFIP, Sigma, St. Louis, MO, USA) to a concentration of 1 mM. For the aggregation protocol, the peptide was resuspended in dry dimethylsulfoxide (5 mM; Sigma, St. Louis, MO, USA). Hams F-12 (PromoCell, LabClinics, Barcelona, Spain) or HCl (10 mM) was added to adjust the final peptide concentration to 100 μM to obtain oligomers (4 °C for 24 h) or fibrils (37 °C for 24 h), respectively. Monomeric Aβ was dissolved in PBS to a concentration of 100 μM. In all experiments Aβ refers to oligomeric amyloid-β (Aβ1–42) unless otherwise stated.
5
Melamine-Assisted Fabrication of Flower-Shaped Manganese Ferrite Nanoparticles
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The flower-shaped NPs B were prepared using the clustering agent melamine. In this method, the reagents N,N′-carbonyldiimidazole (CDI) from FluoroChem (Derbyshire, UK), melamine and imidazole from Sigma-Aldrich (St. Louis, MO, USA) were used [5 (link),6 (link)].
First, manganese ferrite NPs were obtained as previously described [9 (link)]. Then, the −OH groups of the NPs surface were activated before the addition of the clustering agent. For that, 4.3 × 10−5 mol of the prepared manganese ferrite NPs were dispersed in 7 mL of dry dimethyl sulfoxide (from Sigma-Aldrich, St. Louis, MO, USA). Then, 2.2 × 10−4 mol of CDI was added, and the solution was kept at 60 °C, under sonication. After 2 h, ultrapure water was used to eliminate the excess of CDI, and then, 4.3 × 10−5 mol of melamine was added. Finally, an equivalent quantity of imidazole (4.3 × 10−5 mol) was added, and the reaction was kept at 60 °C, for 2 h, under sonication. imidazole was used to promote a faster coupling between the –NH2 groups of melamine and the –OH groups on NPs surface (Figure 3 ) [19 (link),20 (link)].
First, manganese ferrite NPs were obtained as previously described [9 (link)]. Then, the −OH groups of the NPs surface were activated before the addition of the clustering agent. For that, 4.3 × 10−5 mol of the prepared manganese ferrite NPs were dispersed in 7 mL of dry dimethyl sulfoxide (from Sigma-Aldrich, St. Louis, MO, USA). Then, 2.2 × 10−4 mol of CDI was added, and the solution was kept at 60 °C, under sonication. After 2 h, ultrapure water was used to eliminate the excess of CDI, and then, 4.3 × 10−5 mol of melamine was added. Finally, an equivalent quantity of imidazole (4.3 × 10−5 mol) was added, and the reaction was kept at 60 °C, for 2 h, under sonication. imidazole was used to promote a faster coupling between the –NH2 groups of melamine and the –OH groups on NPs surface (
6
Synthesis of Hybrid Biomaterials
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Laminarin
(LAM) from Eisenia bicyclis (CAS: 9008–22–4)
and lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate
(LAP) were purchased from Biosynth. Tetraethyl orthosilicate (TEOS,
98%), hexadecyltrimethylammonium bromide (CTAB, 98%), calcium
chloride anhydrous, dry dimethyl sulfoxide (DMSO, p.a. ≤ 0.02%
water), and formaldehyde were acquired from Sigma-Aldrich. 3-(Trimethoxysilyl)propyl
acrylate (stabilized with BHT) (TMOS-PA, 93%) and glycidyl methacrylate
(stabilized with MEHQ, 95%) were purchased from TCI Chemicals. Disodium
hydrogen phosphate monohydrate and alginic acid sodium salt (Mw 10,000–600 000 Da, Alginate)
were bought from PanReac AppliChem. 4-Dimethylaminopyridine (DMAP,
99%) and toluene (extra dry, 99.85%) were obtained from ACROS Organics.
Unless otherwise specified, all chemicals were used as received without
further purification.
(LAM) from Eisenia bicyclis (CAS: 9008–22–4)
and lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate
(LAP) were purchased from Biosynth. Tetraethyl orthosilicate (TEOS,
98%), hexadecyltrimethylammonium bromide (CTAB, 98%), calcium
chloride anhydrous, dry dimethyl sulfoxide (DMSO, p.a. ≤ 0.02%
water), and formaldehyde were acquired from Sigma-Aldrich. 3-(Trimethoxysilyl)propyl
acrylate (stabilized with BHT) (TMOS-PA, 93%) and glycidyl methacrylate
(stabilized with MEHQ, 95%) were purchased from TCI Chemicals. Disodium
hydrogen phosphate monohydrate and alginic acid sodium salt (Mw 10,000–600 000 Da, Alginate)
were bought from PanReac AppliChem. 4-Dimethylaminopyridine (DMAP,
99%) and toluene (extra dry, 99.85%) were obtained from ACROS Organics.
Unless otherwise specified, all chemicals were used as received without
further purification.
7
Fluorescent Dye Labeling Protocol
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AlexaFluor dyes were purchased
from Thermo
Fisher and used without further purification. Atto 425 dye, dry dimethyl
sulfoxide (DMSO), bisphenol A diglycidyl ether, and triethylenetetramine
were purchased from Sigma-Aldrich and used without further purification.
from Thermo
Fisher and used without further purification. Atto 425 dye, dry dimethyl
sulfoxide (DMSO), bisphenol A diglycidyl ether, and triethylenetetramine
were purchased from Sigma-Aldrich and used without further purification.
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