An overnight LB EPEC culture was diluted 1:50 (vol/vol) with pre-equilibrated (37°C, 5% CO2, 95% humidity) Dulbecco's modified eagle medium (DMEM)-high glucose for 3 hours in a CO2 incubator. When effector protein expression had to be induced, isopropyl β-D-1 thiogalactopyranoside (IPTG; Promega no. V395D; 0.2 mM, unless otherwise indicated) was added to the activation medium after 2.5 hours of incubation. HeLa cells were cultured to 70%–80% confluency on a 24-well plate (NUNC-Cell-Culture Treated Multidishes; no. 142475; Thermo Scientific) or 6-well plate (NUNC-Cell-Culture Treated Multidishes), placed on a heating block (37°C), and washed three times with pre-warmed (37°C) plain DMEM. After the last wash, the medium was replaced with the pre-activated EPEC-containing medium and incubated in the CO2 incubator for the indicated infection times.
Nunc cell culture treated multidishes
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc™ Cell-Culture Treated Multidishes are a line of sterile, single-use cell culture plates designed for in vitro cell growth and assays. The dishes feature a treated surface to promote cell attachment and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Macrophage colony stimulating factor 1, by Thermo Fisher Scientific (1 mentions)
Lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Ultra low attachment flask, by Corning (1 mentions)
Puberogen, by Novartis (1 mentions)
Mitotracker orange cmtmros, by Thermo Fisher Scientific (1 mentions)
Nis elements ar analysis software, by Nikon (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Embryomax m2 medium, by Merck Group (1 mentions)
Leibowitz l 15 medium, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cc 3171, by Lonza (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Promega (1 mentions)
Evos floid cell imaging station, by Thermo Fisher Scientific (1 mentions)
H6024, by Merck Group (1 mentions)
Adipogenic induction medium, by Lonza (1 mentions)
Oil red o, by Muto Pure Chemicals (1 mentions)
Hemocytometer, by Hausser Scientific (1 mentions)
L 428, by Thermo Fisher Scientific (1 mentions)
14 protocols using nunc cell culture treated multidishes
1
EPEC Infection Assay in HeLa Cells
2
Macrophage Polarization Assay with LPS and ONO-1301
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow cells collected from the femurs of 10–12-week-old C57BL/6 male mice were cultured at 37 °C in the presence of 5% CO2 in ultra-low attachment flasks (Corning Inc., Corning, NY, USA) in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Thermo Fisher Scientific, Waltham, MA, USA) containing 20 ng/mL macrophage colony stimulating factor-1 (Peprotech Inc., Rocky Hill, NJ, USA); the medium was changed twice weekly, as described previously [22 (link), 23 (link)]. After 7 days, the collected macrophages were harvested and seeded in 6-well Nunc™ Cell-Culture Treated Multidishes (Thermo Fisher Scientific) at a density of 3 × 105 cells/well. Then, 25 ng/mL lipopolysaccharide (LPS from Escherichia coli O111:B4; catalog number L2630; Sigma-Aldrich, Tokyo, Japan) and 0.01 μM ONO-1301 or DMSO (control group) were added to the cultured macrophages. After 18 h, the macrophages were harvested, and the mRNA expression levels of genes encoding pro-inflammatory factors (e.g., interleukin-6 [Il6], tumor necrosis factor [Tnf-a], monocyte chemotactic protein-1 [Mcp-1], and inducible nitric oxide synthase [Inos]) and anti-inflammatory factors (e.g., interleukin-10 [Il10], chitinase 3-like 3 [Ym-1], and macrophage mannose receptor [Cd206]) were evaluated using real-time polymerase chain reaction (PCR) (Supplementary Table 1 ).
3
Porcine Follicular Oocyte Maturation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The collection of porcine follicular oocytes and in vitro maturation (IVM) were performed as described by Kikuchi et al [24 (link)]. In brief, porcine ovaries were collected at a local slaughterhouse and transported to the laboratory at 37.5°C. Cumulus oocyte complexes (COCs) were collected from 2–6 mm in diameter follicles. Fifty COCs were cultured for 22 h in four-well dishes (Nunc™ Cell-Culture Treated Multidishes; Thermo Fisher Scientific Inc., Waltham, MA, USA), each containing 500 μL of a modified North Carolina State University-37 (NCSU-37) solution [25 (link)] which contained 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 20 μM beta-mercaptoethanol, 1 mM dibutyryl cAMP (dbcAMP), 10 IU/mL eCG (1000 units; PMS; Nippon Zenyaku Kogyo, Fukushima, Japan), and 10 IU/mL hCG (3000 units; Puberogen; Novartis Animal Health, Tokyo). The COCs were subsequently cultured for 22 h in NCSU-37 solution without dbcAMP, eCG or hCG. The maturation culture was performed under 5% CO2 in air at 38.5°C.
4
Immunofluorescent Staining of BV-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BV-2 cells were seeded on cover glasses at a density of 2.0×104 cells/well (Nunc™ Cell-Culture Treated Multidishes, Thermo Fisher Scientific, Waltham, MA, USA). After chemical treatment, cells were fixed with 4% paraformaldehyde/PBS (-) for 15 min at room temperature and were then permeabilized with 0.1% Triton X-100/PBS (-) for 15 min at room temperature. After blocking with 1% BSA/PBS (-) for 60 min at room temperature, cells were incubated with the primary antibody at 4°C overnight. Cells were incubated with the secondary antibody for 2 hours at room temperature, and nuclei were then stained with DAPI for 15 min at room temperature. Cells on the cover glasses were mounted with VECTASHIELD® Mounting Medium (VECTOR LABORATORIES, Burlingame, CA, USA). All steps were carried out in the dark and cells were washed 3 times with PBS (-) or 0.1% Tween 20/PBS (-) after incubation. The antibodies and dilution ratios used in this study are shown in S2 Table . Images were acquired using a confocal laser scanning microscope and analyzed with NIS-Elements AR Analysis software (Nikon, Tokyo, Japan). To label mitochondria, cells were stained with MitoTracker Orange® CMTMRos (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions.
5
Preimplantation Embryo Development in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were pathogen-free, and bred and maintained in the animal care facility at KU Leuven. Animals had access to food (ssniff® R/M-H) and water (HCl-acidified water (pH 2.5-3) and were group-housed in ventilated cages under controlled temperature (22 ± 2 °C) and humidity (45–70%) with a 14 h light, 10 h dark light cycle. To obtain preimplantation embryos, CD-1 female mice were superovulated (SO) by intraperitoneal injection of 150 μL pregnant mare’s serum (PMS) gonadotropin, followed by injection of 150 μL human chorionic gonadotropin (hCG) 48 h later. SO females were then mated with male mice. For β-CATENIN quantification, E3.5 and E4.5 embryos were flushed from dissected uteri with EmbryoMax® M2 Medium (Sigma). Embryos were washed with PBS and fixed in 4% paraformaldehyde (PFA). For ex vivo embryo culture and treatment, E2.5 embryos were flushed from dissected oviducts using EmbryoMax® M2 Medium. E2.5 embryos were treated with 100 ng/mL DKK1 and 10 μM iCRT3 diluted in EmbryoMax® KSOM Medium and cultured in cell culture dish (Nunc™ Cell-Culture Treated Multidishes, Thermo Fisher Scientific) at 37 °C in incubator supplied with 5% CO2. Blastocysts (E2.5 + 48H) were stopped and fixed in 4% PFA for further molecular analysis (see Immunofluorescence staining section).
6
Tankyrase Inhibitor Assay in Colon Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The colon carcinoma cells were seeded (100,000 cells/well) in 6-well plates (Nunc™ Cell-Culture Treated Multidishes, Thermo Fisher Scientific). RPMI1640 were used for HCT15 and COLO320DM (ATCC) cells with incubation in 5% CO2, and Leibowitz L‐15 medium (Thermo Fisher Scientific) for SW480 (ATCC) cells with incubation in 0% CO2. After 24 h, the medium was removed and the tankyrase inhibitor G007-LK [17 (link)] (dissolved in dimethylsulfoxide (Sigma Aldrich) was added to a final concentration of 1 μM in the cells’ respective medium. An equal volume of dimethylsulfoxide was added as negative control. After 24 h of incubation the cells were harvested and processed as described above.
Three biological replicates were made and analyzed, with exception of COLO320DM and SW480 cells, were treated cells were analyzed in duplicates.
Three biological replicates were made and analyzed, with exception of COLO320DM and SW480 cells, were treated cells were analyzed in duplicates.
7
Chrysopidae Specimen Collection and Rearing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sixty-four adults of M. desjardinsi (Insecta; Neuroptera; Chrysopidae) were collected by a sweeping net under street lamps near trees and bushes in the campus of Chiba University, Matsudo, Chiba Pref., Japan, at night (20:00–22:00) from May to October in 2011. M. desjardinsi is not an endangered or protected species. Specific permission is not required for insect collection for students and faculty members in the campus of Chiba University. Females were brought into the laboratory and individually allowed to lay eggs in plastic containers for 15 days. During egg collection, females were fed with 50% honey solution and dried yeast. After egg collection, females were stored at −40°C until DNA extraction. To prevent cannibalism, an egg was placed in each well of the 24-well plate (cat. no., 142475, Nunc™ Cell-Culture Treated Multidishes, Thermo Fisher Scientific K.K., Yokohama, Japan) together with Ephestia kuehniella (Lepidoptera; Pyralidae; Agrisect Inc., Ibaraki, Japan) eggs, as larval diet. Insects were reared under a 16-h:8-h light:dark photoperiod at 25 ± 1°C. Adults were sexed according to the abdominal tip morphology.
8
BEAS-2B Cell Culture Protocol
9
Transfection of HEL and K562 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HEL cell line was a gift from former Professor Marcin Majka from the Jagiellonian University School of Medicine and kept in our laboratory since 2010, as were K562 cells that were a kind gift from Professor D. Duś from the Institute of Immunology, Polish Academy of Sciences in Wrocław, Poland. The cell lines were regularly checked for Mycoplasma contamination. Before transfection, the HEL or K562 cells were grown directly on cover slips for 8 h in 6-well, 35 mm plates (Nunc Cell-Culture Treated Multidishes, Thermo Fisher Scientific) in RPMI 1640 and IMEM medium, respectively, with 10% FCS and antibiotics (100 I.U./mL streptomycin and 100 μg/mL penicillin), then transferred to medium without antibiotics. The cells were transfected with a Lipofectamine 2000 transfection reagent (Thermo Fisher) (μL) to DNA (μg) ratio of 3:1 mixture and cultured. Fluorescent microscopy assessed the changes in morphology resulting from the overexpression of WT or mutated ZZUD fragments after 48 and 72 h (Zeiss AXIO).
10
Immunofluorescence Microscopy of 11β-HSD1/2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence microscopy was performed as previously described (Cirillo et al, 2007 (link)), but with minor modifications. Briefly, cells were cultured in four-well-plates (Nunc Cell-Culture Treated Multidishes, Thermo Fisher Scientific, Paisley, UK; Fisher Scientific UK Ltd, Loughborough, UK) in standard conditions. At 60% confluence, cells were fixed with 100% vol./vol. ice-cold methanol for 10 min at 4 °C and then incubated with 0.5 μg ml−1 Hoetstch staining (H6024, Sigma-Aldrich) for 30 min at 4 °C and then blocked with 5% wt/vol. BSA–PBS solution at 4 °C for 60 min. Samples were incubated overnight at 4 °C with anti-11β-HSD1/2 primary antibody at 1/100 dilution in 2% wt/vol BSA–PBS followed by FITC-conjugated species-specific IgG for 1 h at 1/1000 dilution in 2% wt/vol. BSA–PBS. All intermediate washing steps were performed with PBS. Images were taken with a fluorescence microscope (EVOS FLoid Cell Imaging Station, Life Technologies, Cramlington, UK).
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!