To perform the hemagglutination assay, which allowed fimbriae to be detected, the methodology of Kikuchi et al.37 (link) was applied. P. gingivalis and A. actinomycetemcomitans colonies that had been grown for five days and 24 h, respectively, were suspended in PBS buffer (Sigma) pH 7.4 and adjusted to an absorbance of 2.0 at 660 nm. The oleoresins and isolated compounds were tested at subinhibitory concentrations (½ MIC). To this end, 150 μL of an oleoresin or isolated compound was mixed with 150 μL of the bacterial suspension, which was followed by incubation at appropriate temperature and atmosphere for each strain (anaerobic chamber at 36 °C for P. gingivalis strains and CO2 chamber at 36 °C for A. actinomycetemcomitans strains) in sterile Eppendorf tubes. After incubation, the bacteria were centrifuged at 6000× g for 5 min, resuspended with 500 μL of PBS buffer (Sigma) pH 7.4, and diluted to 1/32 in 96-well plates. A 50-μL aliquot of each dilution was homogenized with 50 μL of a 2% fresh red blood cell suspension in PBS (Sigma) pH 7.4 and incubated in appropriate atmosphere for 3 h. Hemagglutination was visually assessed after incubation.
Pbs buffer
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Hungary, Poland, Italy, Sao Tome and Principe
PBS (Phosphate-Buffered Saline) is a commonly used buffer solution in various laboratory applications. It is a balanced salt solution that maintains a stable pH and osmolarity, providing an optimal environment for biological samples and experiments. PBS is composed of a combination of sodium phosphate and sodium chloride, and its core function is to maintain the physiological conditions necessary for the preservation and study of cells, tissues, and biomolecules.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Formic acid, by Merck Group (4 mentions)
Nile red, by Merck Group (3 mentions)
Sodium hydroxide (naoh), by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Mgcl2, by Merck Group (3 mentions)
Polysine coated microscope slide, by Avantor (2 mentions)
Mouse anti human involucrin sy5, by Abcam (2 mentions)
Alexa fluor 488 labeled goat anti mouse igg antibody, by Abcam (2 mentions)
Dl dithiothreitol, by Merck Group (2 mentions)
Yeast extract, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Avanti mini extruder, by Avanti Polar Lipids (2 mentions)
L glutamine, by Merck Group (2 mentions)
Milli q system, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Trypsin, by Merck Group (2 mentions)
Silver nitrate, by Merck Group (2 mentions)
Prism 8, by GraphPad (2 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (2 mentions)
93 protocols using pbs buffer
1
Hemagglutination Assay for Fimbriae Detection
2
Ang2-siRNA loaded magnetic nanoparticle transfection
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A total of 1 mg CMNPs was added to 1 ml PBS buffer (pH 7.4; Sigma-Aldrich), followed by ultrasonic oscillation for 3 min. Subsequently, 2 ml polylysine (diluted in PBS buffer to a concentration as 0.1 mg/ml; Sigma-Aldrich) was added, followed by mixing well and incubation at room temperature for 10 min. An Ang2-siRNA plasmid (Fig. 1 ; Fuzhou Maixin Biotechnology Development Co., Ltd.) was mixed with polylysine-modified CMNPs, to a ratio of 1:1, 1:10, 1:100 or 1:1,000, followed by incubation at room temperature for 1 h. Ang2-CMNPs were transfected into human malignant melanoma cells. Following transfection, the cells were observed using fluorescence microscopy and the transfection efficiency was calculated by cell counting.
3
Preparation of Fluorescent Latex Beads and DNase I
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Red fluorescent carboxylate-modified latex beads (Sigma, Poole, UK; L3280 500-nm and L5405 1-µm diameters; 2.5% w/v aqueous dispersions) were diluted to 0.125% w/v or 0.25% w/v, respectively, with PBS buffer (Sigma, Poole, UK; pH 7.4). All suspensions were incubated at RT for 20 min prior to use. DNase I from bovine pancreas (2000 Kunitz units per mg protein; Sigma, Poole, UK; D4513) was dissolved in PBS buffer containing 5 mM MgCl2, pH 7.4, to give the enzyme concentration of 20 mg/mL and activity 40 k Kunitz units per mL. Small aliquots of the enzyme stock (20 µL) were frozen in liquid nitrogen and stored at −80°C prior to use.
4
Vancomycin-Labeled Bacterial Imaging
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Bacterial strains 10 and 10ΔmapZ were prepared as described in 2.3 and grown until an OD600 of approximately 0.2. Then 1 ml of the bacterial culture was transferred into a sterile 1.5 ml Eppendorf tube and 5 µl vancomycin‐FL (stock: 100 µg/ml; Thermo Fisher Scientific) as well as 5 µl vancomycin (stock 100 µg/ml; Carl Roth) were added. The cultures were vortexed and incubated at 37°C for additional 20 min. Afterward, the cultures were centrifuged for 10 min at 1690 × g at 4°C. The pellets were washed with HBSS buffer (Thermo Fisher Scientific), resuspended in PBS buffer (Sigma‐Aldrich), and fixed in PBS buffer containing 3% formaldehyde. Bacteria were transferred to poly‐l ‐lysine (Sigma‐Aldrich) coated coverslips, mounted with ProLong® Gold Antifade Reagent (Cell Signaling Technology), and stored at 4°C until examination.
Confocal microscopy was performed using a TCS SP5 confocal laser scanning microscope equipped with a 63× 1.40–0.60‐NA oil HCX Plan Apochromat objective (Leica). Image stacks with a z‐distance of 0.13 µm per plane were acquired using a 1‐Airy‐unit pinhole diameter. Images were deconvolved using Huygens® Essential 20.10 (Scientific Volume Imaging). Maximum intensity projections of 3D‐stacks were generated and adjusted identically for brightness and contrast in ImageJ/Fiji (Schindelin et al., 2012 (link)).
Confocal microscopy was performed using a TCS SP5 confocal laser scanning microscope equipped with a 63× 1.40–0.60‐NA oil HCX Plan Apochromat objective (Leica). Image stacks with a z‐distance of 0.13 µm per plane were acquired using a 1‐Airy‐unit pinhole diameter. Images were deconvolved using Huygens® Essential 20.10 (Scientific Volume Imaging). Maximum intensity projections of 3D‐stacks were generated and adjusted identically for brightness and contrast in ImageJ/Fiji (Schindelin et al., 2012 (link)).
5
DNA-Functionalized Surface Characterization
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Sodium acetate (NaAc; p.a., POCH, Poland), magnesium acetate (Mg(Ac)2; p.a., POCH, Poland), potassium persulfate (VWR Chemicals), potassium hydroxide (POCH, Poland), trisodium phosphate (Chempur, Poland), 1× PBS buffer (pH 7.4; Sigma), absolute ethanol (99.8%; POCH, Poland), EDTA (Sigma), tris–EDTA buffer (TE; Sigma), tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Sigma), 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris; Sigma), 6-mercaptohexan-1-ol (MCH; Sigma) were all of high purity, and were used as received. For all the experiments, we used distilled and deionized water with a conductivity of 0.056 μS cm−1 produced by the Hydrolab system. The following oligonucleotides, purchased from MWG-Operon (Eurofins), were used:
• Capture DNA (5′ → 3′): thiol-C6-GCCTTCACAGGGTCCTTTATGT.
• Complementary target DNA (5′ → 3′): ACATAAAGGACCCTGTGAAGGC.
• Capture DNA (5′ → 3′): thiol-C6-GCCTTCACAGGGTCCTTTATGT.
• Complementary target DNA (5′ → 3′): ACATAAAGGACCCTGTGAAGGC.
6
Isolation and Immunostaining of Cornified Envelopes
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Cornified envelopes were isolated from the tape using an established methodology [29 ]. Briefly, half of each tape was extracted with 750 mL of dissociation buffer containing 100 mM Tris-HCl pH 8.0, 5 mM EDTA (ethylenediaminetetraacetic acid), 2% SDS (sodium dodecyl sulphate) and 20 mM DL-dithiothreitol (Sigma Aldrich Dorset, UK). Tapes were extracted in the dissociation buffer for 10 min at 75 °C and centrifuged at room temperature for 10 min at 5000 g. The extracted CEs were washed (three times) in washing buffer: 20 mM Tris-HCl pH 9.0, 5 mM EDTA, 0.2% SDS and 10 mM DL-dithiothreitol and suspended in 1 PBS buffer (Sigma Aldrich Dorset, UK).
Extracted CEs were transferred onto a Polysine-coated microscope slide (5 μL, VWR international Ltd, Leicestershire, UK) for the immunostaining protocol, as previously described [29 ]. This included an overnight incubation in a humidity chamber at 4 °C in the primary monoclonal antibody (1:100, mouse anti-human involucrin SY5, ABCAM, Cambridge, UK). The antibody solution was washed with PBS three times for 5 min before adding the secondary antibody Alexa-Fluor 488-labeled goat anti-mouse IgG antibody (1:200, ABCAM, Cambridge, UK) for 60 min at room temperature (in the dark). The slides were washed with 1 PBS (three times for 5 min) and mounted with 20 μg/mL Nile red (Sigma Aldrich, Dorset, UK) in 75% glycerol solution.
Extracted CEs were transferred onto a Polysine-coated microscope slide (5 μL, VWR international Ltd, Leicestershire, UK) for the immunostaining protocol, as previously described [29 ]. This included an overnight incubation in a humidity chamber at 4 °C in the primary monoclonal antibody (1:100, mouse anti-human involucrin SY5, ABCAM, Cambridge, UK). The antibody solution was washed with PBS three times for 5 min before adding the secondary antibody Alexa-Fluor 488-labeled goat anti-mouse IgG antibody (1:200, ABCAM, Cambridge, UK) for 60 min at room temperature (in the dark). The slides were washed with 1 PBS (three times for 5 min) and mounted with 20 μg/mL Nile red (Sigma Aldrich, Dorset, UK) in 75% glycerol solution.
7
Immunofluorescent Analysis of Cornified Envelopes
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Cornified envelopes were isolated from the tape using an established methodology [16] and processed using a standardised protocol [12] . In summary, half of each tape was extracted with 750 mL of dissociation buffer containing 100 mM Tris-HCl pH 8.0, 5 mM EDTA (ethylenediaminetetraacetic acid), 2 % SDS (sodium dodecyl sulphate) and 20 mM DL-dithiothreitol (Sigma Aldrich Dorset, UK). The tapes were extracted in the dissociation buffer for 10 min at 75 °C and centrifuged at room temperature for 10 min at 5000 g. The extracted CEs were washed three times and suspended in 1 × PBS buffer (Sigma Aldrich Dorset, UK). Extracted CEs were transferred onto a Polysine-coated microscope slide (5 µL, VWR International Ltd, Leicestershire, UK) and incubated overnight in a humidity chamber at 4 °C with a primary monoclonal antibody against involucrin (1:100, mouse anti-human involucrin SY5, ABCAM, Cambridge, UK). The antibody solution was washed with PBS three times for 5 min before adding the secondary antibody Alexa-Fluor 488-labeled goat anti-mouse IgG antibody (1:200, ABCAM, Cambridge, UK) for 60 min at room temperature (in the dark). The slides were washed with 1 × PBS (three times for 5 min) and a cover slip was mounted with 20 µg/mL Nile red (Sigma Aldrich, Dorset, UK) in 75 % glycerol solution (w/w).
8
Lysate Preparation from SK-MEL-28 Cells
9
Campylobacter Bacteriophage Infection Dynamics
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The growth from C. jejuni BA plates incubated overnight at 42°C were collected and emulsified in PBS buffer (Merck Millipore) for the adjustment of inoculum concentrations. Flasks containing 50 ml nutrient broth No. 2 (Oxoid) were inoculated with C. jejuni cells at starting concentrations of approximately 105 or 107 CFU/ml. After inoculation, flasks were sealed with cotton stoppers and placed in anaerobic jars (Oxoid). Microaerobic conditions were introduced using the gas replacement method (John et al., 2011 (link)). Cultures were then incubated in a shaking incubator at 42°C and 100 rpm. After 2 h incubation, bacteriophage CP_F1 was added at an MOI of 2. Samples were collected at 2 h intervals for determination of viable cell counts and phage titers using the Miles and Misra method. Phage samples were treated with 2 μl chloroform (Thermofisher) and centrifuged at 13,000 × g for 5 min. The supernatant was removed and used for titration. The C. jejuni PT14 wild type and knock-out strains were tested in triplicate to ensure statistical certainty. Growth controls without phage addition served as references.
10
MYC Immunohistochemistry Staining Protocol
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Immunohistochemistry (IHC) staining was performed according to Calcagno, et al.[22 (link)] There were two steps in this protocol. A citrate buffer (MERCK-Germany) was prepared before the test, for performing the Antigen Retrieval Protocol, and phosphate buffered saline (PBS) buffer (MERCK-Germany) for slide washing. Tissue sections were de-paraffinized, rehydrated, incubated in hydrogen peroxide, and Antigen Retrieval was performed by microwave heating (Botan-Iran). The tissue was incubated in a digestive enzyme (pepsin enzyme-invitrogen, USA), for five minutes and then Super Block (Scy Tek-USA) was applied. Primary rabbit monoclonal antibody against MYC (dilution1:100-1:200 ready to use PME 415 AA Biocare Medical, USA) was applied on the slides and incubated overnight in a 4°C humidity chamber. Ultra Tek Anti-polyvalent as a secondary antibody was applied for detection, and Ultra Tek HRP was used (ScyTek Laboratories, UltraTek HRP, USA). Diaminobenzidine (DAB) (Scy Tek, USA) was treated as the chromogen and hematoxylin as the counterstain. Regardless of the strength of stain, any color of the nucleus (with/without staining of the cytoplasm) was assumed as positive. If 10% or more of the tumor cells were positive for MYC protein, the patient was considered as MYC+.
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