Cell morphology, size, and membrane integrity were evaluated using a Zeiss Axio Imager Z2 microscope equipped with Plan-Apochromat objectives and an Axiocam MR R3 (ZEISS, Jena, Germany) for image capture. Membrane integrity was assessed by means of the LIVE/DEAD BacLight Bacterial Viability Kit (L7012, ThermoFisher Scientific). E. coli culture broth (OD600 = 1) was pelleted (10,000× g, room temperature, 5 min), washed twice in NaCl (0.85% w/v%), and stained according to the recommendations of the supplier: 3 µL of a dye mixture containing equal volumes of PI and Syto9 per mL of cell suspension, succeeded by incubation in the dark for 15 min. A total of 5 µL of cell suspension was mounted on PBS-agarose pads (1% w/v%. BR0014G, ThermoFisher Scientific; 0710, VWR) for microscopy [38 (link),39 (link)]. Images were analyzed with ZEN 2.3 Pro software (ZEISS). Cell width (w) and length (l) were measured from phase contrast images, using calibrated scale bars obtained from the imaging software. Cell volumes were calculated assuming a cylindrical cell-shape capped with two half-spheres, applying the formula V = π × w2 × (l − w/3)/4 [40 (link)].
Br0014g
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The BR0014G is a laboratory centrifuge designed for general-purpose applications. It features a capacity of up to 4 x 250 mL and a maximum speed of 4,000 RPM. The centrifuge is suitable for a variety of sample preparation and separation tasks.
Automatically generated - may contain errors
Lab products found in correlation
V 660, by Jasco (3 mentions)
Crystal violet, by Merck Group (3 mentions)
A2153, by Merck Group (2 mentions)
Digitonin solution, by Thermo Fisher Scientific (2 mentions)
Axiocam mr r3, by Zeiss (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Zen 2.3 pro software, by Zeiss (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Bacto peptone, by Merck Group (1 mentions)
Bacto agar, by Merck Group (1 mentions)
Bacto yeast extract, by Merck Group (1 mentions)
F8929, by Merck Group (1 mentions)
M5571, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
C57bl 6j male mice, by Charles River Laboratories (1 mentions)
A31573, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
D4540, by Merck Group (1 mentions)
A1 inverted confocal, by Nikon (1 mentions)
17 protocols using br0014g
1
Comprehensive Bacterial Morphology Analysis
2
Norovirus Genotyping from Fecal Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Faecal specimens submitted to the Institute of Environmental Science and Research for norovirus genotyping were used for this study (HDEC ethical approval number 18/NTA/193). To prepare a norovirus suspension, norovirus GII.4[P16] positive samples were pooled and resuspended in phosphate-buffered saline (PBS), pH 7.2 (BR0014G, Thermo Fisher Scientific) to give a 10% w/v suspension (400 mL total volume). The suspension was filtered through a 0.22 µm membrane (566–0020, Thermo Fisher) and concentrated to 8 mL by the addition of polyethylene glycol (PEG) 6000 (81260, Sigma–Aldrich) to give a final PEG concentration of 10%. This was followed by gentle mixing at 4 °C for 2 h, centrifugation at 10,000×g for 30 min, removal of the supernatant and final elution of the pellet in PBS. The suspension was aliquoted and stored at -80 °C until use. The concentration of norovirus in the resuspended concentrate was quantified using reverse transcription-droplet digital PCR (RT-ddPCR) following RNA extraction, as described below.
3
Ubiquitinated Protein Isolation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293T cells (1 × 106) were transfected with 2.5 μg His-ubiquitin25 (link) and 2.5 μg pcDNA3 (Invitrogen, V790-20), pcDNA3 ATG12 or pcDNA3 ATG12[K-] using GeneJuice (Novagen, 70967) according to the manufacturer's protocol. Forty-eight h after transfection, cells were harvested in phosphate-buffered saline (PBS; Thermo Scientific, BR0014G) and pellets were resuspended in UBA buffer (6 M guanidinium HCl, 300 mM NaCl, 50 mM NaH2PO4 pH 8.0, 100 μg/ml N-ethylmaleimide [Sigma, E3876]). Cell lysates were incubated overnight with His-tag Dynabeads (Novex Life Technologies, 1003D) rotating at 4°C. The next day samples were subjected to consecutive washes in UBA, UBB (1:1 mix of UBA and UBC), UBC buffer (300 mM NaCl, 50 mM Na2PO4 pH 8.0) and PBS. For SDS-Page LDS sample buffer (Novex Life Technologies, NP0007) was added containing 50 mM imidazole.
4
Antifungal Drug Resistance Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The isolates used are listed in Table S1 . The isolates were maintained on YPD agar (1% Bacto Yeast Extract [212750, Sigma], 2% Bacto Peptone [211677, Sigma], 2% Bacto Agar [214010, Sigma], 2% D-[+]-Glucose [G8270, Sigma]), and liquid cultures were grown in 5 mL YPD broth without agar at 30°C and 200 rpm shaking overnight. For serial dilutions, 0.5 mL of the overnight cultures were harvested at 13,000 rpm at room temperature for 1 min, washed twice in 0.5 mL of phosphate-buffered saline (PBS) buffer (BR0014G, Thermo Fisher), resuspended in 0.5 mL PBS, and diluted to A600 = 0.0625 (approximately 6.25 × 105 cells/mL) in PBS. Five-fold serial dilutions were made in PBS and transferred with a pinner to YPD agar plates containing miltefosine (M5571, Sigma-Aldrich) or fluconazole (F8929, Sigma-Aldrich) at the indicated concentrations. The pinned plates were incubated at 30°C for the indicated times and photographed using a Singer PhenoBooth.
5
Rat Brain Inflammation Biomarkers Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On PPD day 10, rats were euthanized using a guillotine, their brains were collected as described previously.8 ,18 Briefly, the brain was dissected out carefully and washed with cold 1x phosphate-buffered saline (PBS) (BR0014G, Oxoid Ltd, UK), 1 tablet dissolved in 100 mL deionized water. The rats prefrontal cortex and hippocampus were carefully harvested, weighted and homogenized using Polytron PT-MR 1600 E (Kinematica AG, Switzerland)with cold PBS (100 mg wet tissue in 1 mL of 1 x PBS) for 3 min. The homogenates were freeze-thawed for 2 cycles to further break down the cells and were centrifuged for 5 min at 5000×g, 4 °C. The supernatants were pipetted and used for the analysis of NFкB, NLRP3, caspase-1 and IL-1β levels, all the biomarkers were measured using their respective ELISA kits according to the user manuals; pro-inflammatory cytokines interleukin-1 Beta (IL-1β) (Rat Il-1β/IL-IF2, E-EL-0012, Elabscience, USA), inflammatory transcription factor, Nuclear Factor Kappa B (NF-kB) (Rat NF-кB, E-EL-0673, Elabscience, USA), inflammasome complex (caspase-1 (Rat CASP1, E-EL-0371, Elabscience, USA) and NACHT, LRR and PYD Domains- Containing Protein 3 (Rat NLRP3, E-EL-1463, Elabscience, USA) which is associated with the production of IL-1β.
6
Intranasal and Intravenous Infection of C57BL/6J Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C57BL/6J male mice, aged 8 to 12 weeks, were purchased from Charles River Laboratories, UK, and infected with 5 × 105 CFU, unless otherwise specified, in sterile phosphate buffered saline (PBS, Oxoid, BR0014G). Intranasal infections were performed by placing 25 µL of yeast suspension into the mouse nares under isoflurane anesthesia. Intravenous infection was performed with 5 × 105 CFU in 100 µL via tail vein injection. Mice were monitored every 6 h for the first 24 h, and then daily, for deterioration in health. We also imaged noninfected (sentinel) mice for tissue morphology, immunolabel, and dye-staining controls, including for CFW and antibody staining specificity. CX3Cr1GFP/+ mice were obtained from University of Exeter colony, a kind gift from Jon Witton and Peter C. Cook.
7
Quantifying Yeast Viability via ATP Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amount of metabolically active yeast cells in cultures after incubation, that is, microbial cell viability, was determined indirectly by measuring adenosine triphosphate with BacTiter GLO™ Microbial Cell Viability Assay (BTG; G8231/G8232). The assay was performed using the Beckman Coulter Biomek NXP and SCARA robotic system. Supernatant (100 μl) was harvested from assay plates and optical density (600 nm) was measured in a microplate reader (SpectraMax® Paradigm® Multi‐Mode Detection Platform). The remaining supernatant in the assay plates was carefully removed from the wells and discarded. Plates were washed once by the robot with 100 μl sterile phosphate‐buffered saline (PBS, BR0014G; Oxoid) and BTG reagent (100 μl) was added. Plates were shaken at 2 g (900 rpm, with 2 mm amplitude) for 30 s on a Quantifoil Instruments GmbH BioShake 3000 elm and incubated in the dark for 15 min. The luminescence was measured in a microplate reader (SpectraMax Paradigm Multi‐Mode Detection Platform). The minimum biofilm inhibitory concentration (MBIC) was defined as the concentration at which growth (BTG‐signal) in all parallels (n = 4) was reduced by 70% compared with the average of controls in the same plate.
8
Immunohistochemistry in Fixed Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixed embryos were serially rehydrated from 100% methanol into PBST [0.2% Tween-20 (Sigma P2287) in 1× PBS (Oxoid BR0014G)], washed three times in 0.2% Triton-X (Sigma T8787) in 1× PBS (PBSTx) and then incubated in blocking buffer for 1 h at room temperature [5% sheep serum (Gibco, 16070-096), 10 mg/ml bovine serum albumin (A2153, Sigma) and 1% DMSO (D4540, Sigma) in PBSTx] for 1 h at room temperature before an overnight incubation at 4°C with gentle agitation with the following primary antibodies: mouse anti-acetylated tubulin (T6793, Sigma, 1:500) and rabbit anti-PKC ζ (aPKC) (sc-216, Santa Cruz, 1:400) in blocking buffer (Amack et al., 2007 (link)). The next day, embryos were rinsed extensively in PBSTx, before a further overnight incubation at 4°C with gentle agitation with the following secondaries: Alexa488-conjugated donkey anti-mouse (Invitrogen A21202, 1:200) and Alexa647-conjugated donkey anti-rabbit (Invitrogen A31573, 1:200) in blocking buffer. Embryos were washed extensively on day three before dissection or embedding. Representative images of Kupffer's vesicle were taken using a Nikon A1 inverted confocal using a 40× objective.
9
Oral Infection of Mice with Listeria monocytogenes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
L. monocytogenes serotype 4a strains acquired from American Type Culture Collection (ATCC 19114), (1010 CFU/mL) were used to study mouse oral infection. Activated culture of L. monocytogenes ATCC 19114 was grown on Brain Heart Infusion Agar (Oxoid, CM 1136) and incubated at 37 °C for 24 h. One colony was picked and inoculated in Brain Heart Infusion broth (Oxoid, CM 1135), for 7 h on rotary shaker (120 RPM) at 37 °C. Optical density (O.D) at 620 nm was determined every 1 h, and at the same time, the total count was enumerated on Brain Heart Agar. After determining the cells of 1010 CFU/mL, (Figures S1 and S2 (supplementary materials) ), the cells were centrifuged (5000 rpm for 10 min); the supernatant was discarded, and the pellet was washed twice in PBS (Dulbecco A, Oxoid BR0014G, pH 7.3) and then resuspended in 10 mL of PBS. Infection dose of L. monocytogenes contained above 109 CFU, and was orally injected to each mouse according to Angelakopoulos et al., 2002; [21 (link)] and Lecuit et al., 1999 [22 (link)]. While, the dose used by Golnazarian et al., 1989 [23 (link)] ranged from 3.74 to 6.45 log10 CFU, we challenged mice orally with 1010 CFU/daily using gavage.
10
Biomass Quantification of Bacterial Biofilms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the validation of the model, six coupons from the third MTP used for biofilm formation and three blank control coupons from the second MTP were used for quantification of biofilm formation based on biomass. 10 mL of a 0.1% crystal violet solution (containing 0.1 g/100 mL crystal violet (Merck, 101,418, Darmstadt, Germany) dissolved in one part of methanol (Biosolve, 13,687,802, CE Valkenswaard, The Netherlands), one part of isopropanol (Merck, 1.09634) and 18 parts of Phosphate Buffered Saline (Oxoid, BR0014G)) was added to each of the falcon tubes for 20 min and shaken (Fisher Bioblock Scientific, KL2 6118 CU 00246, Merelbeke, Belgium) at 350 rpm for the staining of the total biomass of the biofilm on the coupons. The excess stain was removed by placing the tubes under gently running tap water. Retained crystal violet was dissolved by adding 10 mL of 33% acetic acid (Merck, 1.00063) for 15 min at 350rmp. The absorbance was measured at 590 nm using a spectrophotometer (Jasco, V-660, Pfungstadt, Germany). OD-measurements of the blank control coupons were subtracted from the OD-measurements of the biofilm coupons.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!