The following components were used for the Quantitative reverse transcription Polymerase Chain Reaction (RT-qPCR): primer mix (10 pmol/μL) 1 μL, 2 μM STYO 9 (ThermoFisher), 1X THUNDERBIRD SYBR RT-qPCR mix (TOYOBO) and 2.5 mM Mn(OAC)2. The detailed reaction protocol was as follows: pre-denaturation at 95 °C for 30 s, reverse transcription at 61 °C for 20 min, pre-denaturation at 95 °C for 1 min, followed by 45 cycles consisting of denaturation step at 95 °C for 15 s, annealing at 58 °C for 15 s, and extension at 74 °C for 30 s. Serial diluents of SARS-CoV-2 RNA (NR-52499, BEI resource) were used as a calibration curve for quantification, and the RNA copy number in each sample was calculated using the following formula Eq. (1) .
Styo 9
Manufactured by Thermo Fisher Scientific
Sourced in United States
The STYO 9 is a laboratory instrument designed for the fluorescent detection and quantification of nucleic acids. It functions as a sensitive and selective dye that binds to double-stranded DNA, allowing for accurate measurement of DNA concentration in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Thunderbird sybr rt qpcr mix, by Toyobo (1 mentions)
Isothermal amplification buffer, by New England Biolabs (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Temozolomide, by Meilun (1 mentions)
Fv1200, by Olympus (1 mentions)
Glass bottomed cell culture dish, by NEST Biotechnology (1 mentions)
5 protocols using styo 9
1
SARS-CoV-2 RNA Quantification by RT-qPCR
2
Quantitative RT-LAMP for Molecular Diagnostics
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The following components were used for the Quantitative reverse transcription Loop-Mediated Isothermal amplification: 2 μM STYO 9 (ThermoFisher), 1 X isothermal amplification buffer (New England Biolabs), 1.4 mM deoxyribonuclease triphosphates (New England Biolabs), 8 U of BST 2.0 Warmstart DNA polymerase, 4 U of RTx. Reverse transcriptase, 0.2 μM F3 and B3, 1.6 μM FIP and BIP, and 0.4 μM LF and LB. The detailed reaction protocol was as follows: 90 cycles consisting of reverse transcription and extension at 60 °C for 1min.
3
Bacterial Culture Media Preparation
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LB broth and nutrient agar were made up from BactoTM Tryptone Pancreatic Digest of Casein and BactoTM Yeast Extract, purchased from Bacto Laboratories Pty, Ltd., (Mt Pritchard, NSW 2170, Australia), and NaCl and Agar A bacteriology Grade purchased from Astral Scientific (Taren Point, NSW 2229, Australia). Selective agars Xylose Lysine Deoxycholate and Eosin Methylene Blue were purchased premade from Edwards (Narellan, NSW, Australia). Staphylococcus aureus ATCC 12600TM, Salmonella typhimurium ATCC 14028TM, and Escherichia coli ATCC 43888TM were purchased from Cell Biosciences (Victoria, Australia). STYO 9 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). DAPI was purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
TMZ-Loaded Nanoparticle Delivery System
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Temozolomide (TMZ) was acquired from Dalian MeiLun Biotechnology Co., Ltd. RVG29-Cys peptide (sequence: N term-YTIWMPENPRPGTPCDIFTNSRGKRASNGC-Cys) was synthesized by American Peptide Company (Sunnyvale, CA, USA). Phenazine methosulfate (PMS), dimethyl sulfoxide, Zein, coumarin-6 (C6), and paraformaldehyde (PFA) were acquired from Hongquan Biotechnology Co., Ltd. All remaining reagents, if not specified, were purchased from Shanghai Titan Science Co. (Shanghai, China). Live/Dead cell imaging kits, STYO 9, and propidium iodide (PI) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ELISA kits were bought from Shanghai Yuanxin Biotechnology Co., Ltd. Fetal bovine serum (FBS), Phosphate buffered saline, Dulbecco's modified Eagle's medium (DMEM), Tyrisin and l -glutamine were provided by Gibco. Cleaved caspase-3 and Caspase-3 antibodies were bought from Dojindo Laboratories (Kumamoto, Japan). Annexin V-FITC/PI apoptosis detection kit and D-luciferin were supplied by Shanghai Yeasen Corporation. All other chemicals were available from commercial sources and used without further purification.
5
Assessing TPCP's Effect on Biofilm Viability
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SYTO9 green fluorescent nucleic acid staining was used to assess the effect of TPCP on the viability of MSSA and MRSA biofilm (Jefferson et al., 2005 (link)). Bacterial suspension (2 mL, 1.5 × 108 CFU/mL) was added to a glass-bottomed cell culture dish (NEST, Wuxi, China) and incubated for at 35°C 72 h. The planktonic bacteria were then removed, and 500 μL of fresh MHB containing different final concentrations of TPCP (2×, 1×, and 1/2 × MIC) were added. A control group was set up separately. After 8 h of incubation, the supernatant was discarded and the cells were washed with 5 mM 4-(2-hydroxyethy)-1-piperazine-1-ethanesulfonic acid (HEPES) buffer (pH 7.2). STYO9 (1 μL, Thermo Fisher, USA) was added to each dish and incubated for 20 min, and observed under a CLSM (Olympus FV1200, Japan) after the dye was removed. This experiment was repeated three times.
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