Protamine sulfate salt from salmon

Protamine sulfate salt from salmon (5.1 kDa, p4020, Lot# SLBW4512, Oncorhynchus keta), Poly(ethylene glycol) (10 kDa), Sigmacote (SL2), Particles with a diameter of 2 μm (L3030), Fluorescein-5(6)-isothiocyanate (F3651) and 4-Carboxy-TEMPO (382000) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Hyaluronic acid (5 kDa) was purchased from SK-bioland (Seoul, South Korea) 5(6)-SFX (6-(Fluorescein-5-(and-6)-Carboxamido) Hexanoic Acid, Succinimidyl Ester) (F2181) was purchased from Thermo Fisher (Waltham, MA, USA). 2% PFPE-PEG-PFPE tri-block copolymer surfactant (E2K0660) from RAN Biotechnologies, Inc. (Beverly, MA, USA) were used.

To immobilize giant
vesicles, calcium fluoride slides were pretreated with 10 mg/mL protamine
(Protamine sulfate salt from salmon, Sigma) for 10 min. After 5 washes
with DPBS, 20 μL of vesicle suspension was incubated on slides
for 1 h, before 20 washes with DPBS. Vesicles were imaged using the
alpha 300R+ confocal Raman microscope (Witec GmBH, Germany). A 35
mW, 532 nm laser light source was shone through a 63× 1.0 NA
water immersion objective lens (W Plan-Apochromat, Zeiss, Germany).
Raman scattering was collected through the same lens and directed
via a 100 μm diameter silica fiber to a 600 groove/mm spectrograph
(UHTS 300, WITec, GmbH, Germany) coupled to a back-illuminated charge-coupled
device camera, cooled to −60 °C. Area scans of vesicles
were imaged with 500 × 500 XY nm resolution. Spectral preprocessing
was performed with ProjectFIVE software (Witec GmBH). First, cosmic
rays were removed, and then the dark current background was subtracted,
followed by “shape” background correction. Spectra were
normalized to the maximum intensity of the water peak at 3000–3400
cm^–1. Finally, Raman images were reconstructed from
univariate analysis of the intensity of the NH₂ signal at 2208 cm^–1 and the SO₃ signal at 2220 cm^–1. Due to low intensity of the 708 cm^–1 polymer
peak with this instrumental setup, Raman images of polymer signal
were reconstructed from 2905 cm^–1 polymer peak.

Calf thymus DNA, protamine sulfate salt from salmon, Ethidium bromide, netropsin dihydrochloride and 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma Aldrich Co. and used without further purification. Self-complementary primers for ^GCDNA i.e. 5′-GCGCGCGCGC-3′ and 5′-GCGCGCGCGC-3′ and for ^ATDNA i.e. 5′-GCAAATTTGC-3′ and 5′-GCAAATTTGC-3′ were purchased from Integrated DNA technologies. Their duplexes were prepared by heating the two complementary strands at 85–90 °C, for 5 min, and then slowly cooling to room temperature. The final concentration of the DNA was determined spectrophotometrically by measuring absorbance at 260 nm using a molar extinction coefficient of ε₂₆₀ = 13200 bpM⁻¹ cm⁻¹ for ^CTDNA^{29 (link)}, ε₂₆₀ = 158840.8 M⁻¹ cm⁻¹ for ^GCDNA and ɛ₂₆₀ = 153441.4 M⁻¹ cm⁻¹ per strand for ^ATDNA. The stock solution of 1 mM each of DAPI, Net, EtBr and protamine were prepared in HPLC water.

High Glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM) (# 11965092, Gibco/Thermo Fisher Scientific, Waltham, MA), fetal bovine serum (FBS) (# 16-000-044, Gibco/Thermo Fisher Scientific), calf serum (CS)(#S0400, Biowest), L-glutamin (# 25030081, Gibco/Thermo Fisher Scientific), antibiotic/antimycotic (# 15240062, Gibco/Thermo Fisher Scientific), penicillin/streptomycin (# 15140122, Gibco/Thermo Fisher Scientific), Ficoll-Paque Plus (# 17-1440-03, GE Healthcare, Piscataway, NJ), Puromycin (# P8833, Sigma-Aldrich, St Louis, MO), Protamine sulfate salt from salmon (# P4020, Sigma-Aldrich), Methocel A4M (# 94378, Sigma-Aldrich), Dexamethasone (# D1756, Sigma-Aldrich), 3,3′,5-Triiodo-L-thyronine (T3) (# T6397, Sigma-Aldrich), Troglitazone (# 71750, Cayman Chemical, Ann Arbor, MI), Porcine insulin (# I5523, Sigma-Aldrich), Prostaglandin F2α (#16010, funakoshi, Tokyo, Japan), omidenepag (generous gift from Santen Pharmaceutical Co., Ltd., Osaka, Japan), butaprost free acid (#13741, funakoshi, Tokyo, Japan).