Dulbecco s phosphate buffered saline

The following analytical-grade chemicals were purchased from commercial sources and used without further purification: polyethylene glycol methacrylate (MA-PEG, Mn 360), tetraethylene glycol diacrylate (TTEGDA), polyethylene glycol methacrylate ether (MA-PEG-OCH₃, Mn 300), potassium persulfate (PPS), O-[(N-Succinimidyl) succinyl-aminoethyl-O′-methylpolyethylene glycol (PEG-NHS, Mw 750), polyvinylpyrrolidone (PVP, Mw 360 K), sodium hydroxide (NaOH, 1 N), hydrochloric acid (HCl, 1 N), anhydrous dichloromethane, anhydrous N,N-dimethylformamide (DMF), chromium oxide, isopropanol, magnesium sulfate (97 %), triethylamine (99 %), methanesulfonyl chloride, sodium chloride, sodium azide (99.5 %), triphenylphosphine, glycine and O,O′-Bis[2-(N-succinimidyl-succinylamino)ethyl]polyethylene glycol (NHS-PEG-NHS, MW 3,000) from Sigma (Rehovot, Israel); N-(3-aminopropyl) methacrylamide hydrochloride, (APMA) from Polysciences, Inc. (Warrington, PA, USA) Dialysis membrane (1000 K–16 MM), bicarbonate buffer (BB, 0.1 M, pH 8.4), sodium carbonate and sodium bicarbonate (BB) from Bio-Lab Ltd. (Jerusalem, Israel); Cy 7-NHS ester from Lumiprobe Corporation (Florida, USA); Dulbecco’s phosphate-buffered saline (PBS) from Biological Industries (Bet Haemek, Israel). Water was purified by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga Ltd., High Wycombe, UK).

To determine the toxicity of the peptides to normal mammalian cells, human erythrocytes were used to test the hemolytic activity of the peptides according to the method reported [53 (link)]. Blood was collected from the median cubital vein of healthy volunteers. The normal human erythrocytes were washed three times with Dulbecco’s phosphate-buffered saline (PBS; Biological Industries, Israel), with the resulting pellet was suspended in PBS and diluted to 5% (w/v). The sample peptide was diluted with physiological saline into various gradient concentrations, 200 μL of various gradient concentrations of samples and 200 μL diluted red blood cells were gentle mixed and incubated at 37 °C for 30 min. Parallel incubations of erythrocytes in the presence of normal saline or 1% Triton X-100 served as the negative and positive controls, respectively. After incubation, centrifugation was conducted at 2500 rpm/min for 5 min at room temperature. A total of 100 μL of the supernatant was taken to measure the absorbance at 540 nm (GeneQuant, Fairfield, CT, USA). The relative optical density was compared with the absorbance of 1% Triton X-100, which induce 100% hemolysis. The study was proved by the Research Ethics Committee of Kunming Institute of Botany, Chinese Academy of Sciences and the ethics approval number was Kib202103028. Informed consent was provided for blood donation.

E. coli BL21-DE3 cells expressing the two plasmid systems (a single copy plasmid containing the binding site cassette, and a multicopy plasmid containing the RBP-GFP) were grown overnight in 5 ml LB, at 37 °C with appropriate antibiotics (CM, Amp), and in the presence of two inducers—1.6 μl isopropyl β-D-1-thiogalactopyranoside (IPTG) (final concentration 1 5 mM), and 2.5 μl C4-HSL (final concentration 60 μM) to induce expression of T7 RNA polymerase and the RBP-FP, respectively. Overnight culture was diluted 1:100 into 3 ml solution of (BA-LB (95%–5% v-v) with appropriate antibiotics and induced with 1 μl IPTG (final concentration 1 mM) and 1.5 μl C4-HSL (final concentration 60 μM). For stationary phase tests, cells were diluted into 3 ml Dulbecco’s phosphate-buffered saline (PBS) (Biological Industries, Israel) with similar quantities of induction and 10 antibiotics. Culture was shaken for 3 h in 37 °C before being applied to a gel slide (3 ml PBS×1, mixed with 0.045 g SeaPlaque low melting agarose (Lonza, Switzerland), heated for 20 s and allowed to cool for 25 min). 1.5 μl cell culture was deposited on a gel slide and allowed to settle for an additional 30 min before imaging.

ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), Akt (pan), phospho-Akt (S473), CD79b, CD79a, phospho-CD79a (Tyr182), SHP1, phospho-SHP1 (Tyr564), SHIP1, phospho-SHIP1 (Tyr1020), BTK, phospho-BTK(Tyr223), NF-kB p65, phospho–NF-kB p65 (ser536), IgM, Lyn, phospho-Lyn (Y507), LCK, CD86, and ZAP70 antibodies were from Cell Signaling Technology (Beverly, MA). Anti-SRC family (phospho-Y418)–phospho-Lyn (Y396), CD62L, and IgG antibodies were from Abcam (Cambridge, UK). Purified anti-human actin antibody was obtained from MP Biomedicals (Illkirch, France). Goat anti Rabbit IgG (H+L)–HRP conjugate and Goat anti Mouse IgG (H+L)–HRP conjugate, Goat F(ab′)2 anti-human IgM and Goat F(ab′)2 anti-human IgG were from Jackson Immunoresearch Laboratories (West Grove, PA). All antibodies utilized in the study were used in concentrations according to the manufacturer’s instructions. Ficoll-Paque PLUS from GE healthcare (Uppsala, Sweden), dimethyl sulfoxide (DMSO) from Merck (Darmstadt, Germany), RPMI, fetal calf serum (FCS), Dulbecco’s phosphate-buffered saline (PBS), L-glutamine, and penicillin-streptomycin from Biological Industries (Beit-Haemek, Israel) were utilized for cell cultures.

Dulbecco’s modified Eagle’s medium (DMEM), Leibovitz-15 medium, glutamine, antibiotics (10,000 IU/mL penicillin and 10,000 μg/mL streptomycin), soybean trypsin inhibitor, fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (PBS) (without calcium and magnesium) were purchased from Biological Industries (Beit Haemek, Israel); memantine, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was obtained from Applichem (Darmstadt, Germany); and hydrogen peroxide (H₂O₂) was obtained from MP Biomedicals (Solon, OH, USA).

Silk fibroin was provided by the School of Biotechnology, Jiangsu University of Science and Technology. Chloroauric acid tetrahydrate (HAuCl₄·4H₂O), gadolinium(III) chloride hexahydrate (GdCl₃·6H₂O), chlorpromazine hydrochloride, genistein, and NaOH were obtained from Shanghai Mackin Biochemical Co., Ltd. Wortmannin was purchased from Aladdin (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (PBS) were purchased from Biological Industries. Acridine Orange/Propidium Iodide (AO/PI) stain was obtained from Nexcelom Bioscience. Cell counting kit-8 (CCK-8) was ordered from Yeasen Biotech Co., Ltd., (Shanghai, China). Fluorescein isothiocyanate (FITC) was supplied by Shanghai Mackin Biochemical Co., Ltd., (Shanghai, China). All aqueous solutions used in experiments were prepared with deionized water (18.2 MΩ·cm resistivity at 25 °C).

A piece of skin of 1 cm² was taken from a rat strain Sabra. The skin was maintained in Dulbecco's Phosphate Buffered Saline, PBS (Biological Industries, Israel), while it was divided and cut with a scalpel in 10 pieces. Skin fragments were washed once for 30 min at 4°C with 3 ml of PBS supplemented with 1% of penicillin-streptomycin, three times for 10 min with 3 ml of PBS without antibiotic and once with culture medium as was described before. These skin pieces were cultured in a 12-well plate (2–3 pieces/well). The dermal side of each skin fragment was seeded in touch with the well surface to allow adhesion for 30 s without medium. Finally, 1 mL of culture medium was added carefully to avoid detaching the skin from the well bottom and incubated at 37°C in 5% CO₂. The medium was replaced every 2–3 days. After 1 week, when the wells had become 80–90% confluent, cells were detached with trypsin solution and plated in a tissue culture dish (100 mm) or used for subsequent experiments. Passage numbers 2–4 were used for the experiments.