Observation of S. mutans strains binding to type I collagen using confocal laser scanning microscopy was performed by a method described previously43 (link), with some modifications. The collagen-binding assay described above was performed using a chambered cover glass system (CultureWell™, Grace Bio Labs, Bend, OR, USA) instead of a 96-well plate. After binding the bacteria to collagen for 3 h, bacterial cells were stained with 5 µl of 10 mM hexidium iodide (Invitrogen, Carlsbad, CA, USA) in 1 ml of Hanks’ balanced salt solution (Lonza, Walkersville, MD, USA) for 15 min at room temperature in the dark. Stained bacteria were observed by confocal scanning laser microscopy using a TCS-SP5 microscope (Leica Microsystems GmbH, Wetzlar, Germany) with reflected laser light at 543 nm, as well as a DMI6000 B fluorescence microscope (Leica Microsystems GmbH) and a 63 × oil immersion objective.
Hank s balanced salt solution
Manufactured by Lonza
Sourced in United States, Switzerland, Germany
Hank's Balanced Salt Solution is a sterile, isotonic saline solution commonly used in cell culture applications. It provides a balanced salt composition to support the maintenance and growth of various cell types in vitro. The solution contains inorganic salts, glucose, and phenol red as a pH indicator.
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Lab products found in correlation
Hexidium iodide, by Thermo Fisher Scientific (4 mentions)
Facscalibur, by BD (4 mentions)
Lsm 510, by Zeiss (4 mentions)
Dmi6000b fluorescence microscope, by Leica (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tcs sp5 microscope, by Leica (2 mentions)
Dulbecco s phosphate buffered saline, by Lonza (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Hepes, by Lonza (2 mentions)
Apc conjugated anti cd326 epcam antibody, by Miltenyi Biotec (2 mentions)
Clone ber ep4, by Agilent Technologies (2 mentions)
Ber ep4, by Agilent Technologies (2 mentions)
Facsaria 2, by BD (2 mentions)
Accutase cell detachment solution, by Merck Group (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Gelatin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cts synth a freeze medium, by Thermo Fisher Scientific (1 mentions)
40 protocols using hank s balanced salt solution
1
Visualizing S. mutans Collagen Binding
2
Intraperitoneal Glucose Tolerance Test
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After an overnight fast, the animals received an intraperitoneal glucose bolus (2 g/Kg of body weight) as a 5% solution in Hanks’ Balanced Salt Solution (Lonza), and blood glucose was monitored at 0, 15, 30, 60, 90 and 120 min. To evaluate the response to IPGTTs, the “area under the curve” was calculated for each animal using the trapezoidal rule method [17 (link)].
3
Autologous Tumor Lysate Preparation Protocol
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For the autologous tumor lysate preparation, a cervical punch biopsy was collected in 10 mL of Hank’s balanced salt solution (Lonza, Switzerland) (ice cold) containing 100 IU per mLs of penicillin, streptomycin, and gentamicin (Lonza, Switzerland) each. Sample processing was carried out on the same day of collection and processed in 6 mL of HBSS containing 0.6 Pz units of collagenase (NB6 GMP-grade from Serva GmBH, Heidelberg, Germany) and incubated overnight at 37 °C/5% CO2 to allow for tissue dissociation). Traces of collagenase were removed by washing with calcium-free DPBS. The final cell suspension was passed through a sterile 70 μm cell strainer (Miltenyi MACS, Waltham, MA, USA) to obtain a fine single-cell suspension. Approximately 2 × 105 cells were immobilized on slides and verified by a cytologist to obtain the tumor cell percentage. The remaining cells in the single-cell suspension (SCS) were frozen in a ready-to-use, serum-free, GMP-grade cryopreservation medium with 10% Dimethyl sulfoxide (DMSO) (CTS™ Synth-a-Freeze™ Medium—Gibco™, Waltham, MA, USA) as per the manufacturer’s instructions. The tumor lysates were prepared as described previously [19 (link)] and evaluated for sterility followed by cryopreservation at −80 °C until use for antigen priming.
4
EpCAM and CD90 Cell Identification
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Harvested cells were resuspended in Hank's Balanced Salt Solution (Lonza, Basel, Switzerland) supplemented with 1% HEPES (Gibco) and 2% FBS. Cells were incubated with FITC‐conjugated anti‐EpCAM antibody (Dako) or FITC‐conjugated anti‐CD90 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) on ice for 30 min. Labeled cells were analyzed with a flow cytometer FACSCalibur (BD Biosciences, San Jose, CA, USA). Flow cytometry data were analyzed using flowjo ™ Software V.10 (BD Biosciences).
5
Corneal HSV-1 Infection in BALB/c and B6 Mice
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Female BALB/c and B6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and used at 6–8 weeks of age in all experiments. Mice were anesthetized by intraperitoneal injection of 100 mg/kg body weight ketamine hydrochloride and 0.1 mg/ kg body weight xylazine (Phoenix Scientific, San Marcos, CA, USA) in 0.2 mL Hanks' balanced salt solution (BioWhittaker, Walkersville, MD, USA). Topical corneal infection was performed by scarification of the central cornea with a sterile 30-gauge needle in a crisscross pattern and applying 3 μL RPMI (BioWhittaker) containing 1 × 105 pfu of HSV-1. BALB/c mice were infected with the KOS strain of HSV-1, whereas B6 mice were infected with RE HSV-1. These viral/mouse strain combinations resulted in HSK in 80%–100% of mice. The HSV-1 was grown in Vero cells, and intact virions were purified on OptiPrep density gradients (Accurate Chemical and Scientific Corp., Westbury, NY, USA) and stored at −80°C. Concentration of HSV-1 was determined in a standard virus plaque assay. Experimental procedures were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
6
Isolation and RNA-seq of Tumor Cells
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Primary tumor tissues were minced, and single-cell suspensions were obtained. A detailed procedure is provided in the supplementary methods. The collected cells were incubated with antibody for 30 min on ice as follows: 10 μL anti-EpCAM (R&D Systems, FAB9601F) per 106 cells in 100 μL buffer; 5 μL anti-CD45 (BD, 557748) per 106 cells in 100 μL buffer; 10 μL anti-CD31 (Miltenyi Biotec, 130-092-652) per 107 cells in 100 μL buffer; and 2.5 μg of FAP (R&D systems, MAB3715) per 106 cells in 100 μL buffer. After washing with Hank’s balanced salt solution (Lonza) twice, the cells were incubated with mouse IgG (H + L) PE as follows: 10 μL per 106 cells in 100 μL buffer for 30 min on ice. After washing away the unstained secondary antibody, the cells were resuspended in 1 mL PBS and then sorted using a BD FACSARIA III (BD Biosciences). Total RNA sequencing libraries were prepared according to the manufacturer’s instructions (Illumina TruSeq RNA Access Library kit). The flow cell was then loaded on a HiSeq 2500 sequencing system (Illumina), and sequencing was performed using 2 × 100 bp read lengths.
7
BHK-21 Cell Culture and Virus Infection
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Baby hamster kidney (BHK-21;) and BSR cell monolayers [47, 48] were cultured in 75 cm 2 flasks using BioWhittaker Dulbecco's Modified Eagle's Medium (DMEM) (Lonza), supplemented with 5% (v/v) foetal bovine serum (FBS) (HyClone), 1% (v/v) L-glutamine [200 mM in a 0.85% (w/v) NaCl solution, Lonza] and 1% (v/v) PSA (10 000 U/ml of penicillin, 10 000 µg/ml of streptomycin and 25 µg/ml of amphotericin B; HyClone). The flasks were incubated at 37°C, with 5% CO2, in a humidified incubator.
Cell culture inoculations were prepared by resuspending lyophilized preparations of different strains (Table 1) in Hanks balanced salt solution (Lonza) and infecting BHK-21 monolayers at a confluency of 70-80% with the virus. Monolayers were incubated for 3-4 days, or until cytopathic effects were observed. Infected cell monolayers were used for infections and RNA extraction.
Cell culture inoculations were prepared by resuspending lyophilized preparations of different strains (Table 1) in Hanks balanced salt solution (Lonza) and infecting BHK-21 monolayers at a confluency of 70-80% with the virus. Monolayers were incubated for 3-4 days, or until cytopathic effects were observed. Infected cell monolayers were used for infections and RNA extraction.
8
Isolation and Cryopreservation of pBD-ECs
9
Confocal Microscopy Analysis of Streptococcus mutans Biofilm
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The bacterial biofilm formed on the monofilament was analysed using confocal laser scanning microscopy as described previously31 (link), with some modifications. The S. mutans MT8148 adhering to the monofilaments were resuspended in 1 ml of sterile distilled water including 5 µl of 10 mM Hexidium Iodide (Invitrogen, Carlsbad, CA, USA) and incubated in the dark for 15 min at room temperature. The monofilament was washed with Hanks’ Balanced Salt Solution (Lonza, Walkersville, MD, USA), fixed with 4% paraformaldehyde, and mounted on a slide glass. The bacterial mass of S. mutans on the monofilament was observed by confocal scanning laser microscopy using a TCS-SP5 Microscope (Leica Microsystems GmbH, Wetzlar, Germany) with reflected laser light at 543 nm, as well as a DMI6000 B Fluorescence Microscope (Leica) and a 63 × oil immersion objective.
10
Cellular Bioenergetics and Viability Assay
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Dimethyl sulfoxide (DMSO), Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s Modified Eagle Medium (DMEM), tissue culture grade water, trypsin, and Hanks balanced salt solution (HBSS) were obtained from Lonza (Walkersville, MD, USA). Fluorescent probes, 5,5′, 6,6′-tetrachloro-1,1′, 3,3-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), and propidium iodide (PI) were obtained from Sigma-Aldrich (St Louis, MO, USA). Fetal Bovine Serum (FBS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V and fluorescein conjugate (FITC annexin V) was purchased from Invitrogen (Carlsbad, CA, USA). The oxiselect ™ intracellular ROS Assay Kit (Green Fluorescence) was purchased from Cell Biolabs (San Diego, CA, USA). The XF Cell Mito Stress Kit was purchased from Agilent technologies (Santa Clara, CA, USA).
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