To evaluate clonogenic survival, HCT116 cells were seeded at two different concentrations (100 and 200 cells/well) in 24-well plates. Twenty-four hours later, cells were treated with different concentrations of the Trachinus vipera purified venom (50, 100, 500 and 1000 μg/ml) for one week. Colonies were then fixed/stained with an aqueous solution containing 0.25% (w/v) crystal violet, 70% (v/v) methanol and 3% (v/v) formaldehyde (Carlo Erba Reagents) and counted. Only colonies made of >30 cells were included in the quantification. For each treatment, the survival fraction (SF) was estimated according to the formula: SF = number of colonies formed/number of cells seeded.
Formaldehyde
Manufactured by Carlo Erba
Sourced in France
Formaldehyde is a colorless, flammable gas that is commonly used as a chemical reagent and preservative in laboratory settings. It has a pungent, irritating odor. Formaldehyde is primarily used as a fixative and preservative in the preparation of biological samples for microscopic examination.
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Lab products found in correlation
Crystal violet, by Carlo Erba (1 mentions)
Mica microscope, by Leica (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
3 isobuthyl 1 methylxanthine, by Merck Group (1 mentions)
Dmem f12, by Dutscher (1 mentions)
Sudan black b, by Merck Group (1 mentions)
Sphingomyelin, by Avanti Polar Lipids (1 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by Thermo Fisher Scientific (1 mentions)
1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Paraffin wax, by Carlo Erba (1 mentions)
Dioleoylphosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Dioleoyl phosphatidylglycerol, by Avanti Polar Lipids (1 mentions)
Acetic acid, by Carlo Erba (1 mentions)
Gram s crystal violet solution, by Merck Group (1 mentions)
Rm2265, by Leica (1 mentions)
7 protocols using formaldehyde
1
Clonogenic Assay of Trachinus vipera Venom
2
Evaluation of Cadherin Expression in 3D Spheroids
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Fifty thousand cells/well were seeded into a 6-well plate coated with polyHEMA and the obtained spheroids were collected, transferred into 6-well plates with coverslips on the bottom, and stimulated as above. At 3 and 48 h from stimulation, the cells were fixed with 4% formaldehyde (Carlo Erba Reagents, Emmendingen, Germany) and used in the immunofluorescence evaluation of E-cadherin and N-cadherin markers. Details were reported in the Supplementary Materials . Slides were visualized using a Leica MICA microscope in confocal mode.
3
Adipocyte Differentiation Assay
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For adipocyte differentiation, ASC were amplified until 80% of confluency was reached and culture medium replaced by differentiation medium containing Dulbecco's modified Eagle medium/Nutrient Mixture F12 (DMEM/F12; Dominique Dutscher, France), FBS 10% (Hyclone, France), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 1 μg/ml insulin, 0.5 μM dexamethasone, 2 nM Triiodo-L-Thyronine 3 (T3), 0.5 μM 3-isobuthyl-1-methylxanthine (IBMX), 2 μM rosiglitazone, and 10 μg/ml transferrin, all from Sigma-Aldrich. After 7 days, adipocyte differentiation was measured by Oil red O staining of the neutral triglyceride and lipids. Cells were fixed in 10% formaldehyde (Carlo Erba, France) for 1 hour then washed with 60% isopropanol (Carlo Erba) twice, dried and colored by incubation for 10 minutes with 0.2% Oil red O (Sigma-Aldrich) in isopropanol. Brightfield images were taken with an optic microscope with a ×20 magnification. The expression of adipogenic genes (Pparγ, Dgat2, and Hsl) was measured by RT-qPCR.
4
Lipid Bilayer Preparation and Characterization
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1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylglycerol (DOPG), Sphingomyelin (SM), N-(7-nitro-benz-2-oxa-1,3-diazol-4-yl) phosphatidylethanolamine (NBD-PE) and N-(Lissamine-rhodamine-B-sulfonyl) phosphatidylethanolamine (Rho-PE) were purchased from Avanti Polar Lipids (Birmingham, AL, USA), while cholesterol (CHOL), Triton 100X and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) were purchased from Sigma (St. Louis, MO, USA). DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate) was purchased from Invitrogen (Carlsbad, CA, USA), Sudan Black B from Sigma-Aldrich (St. Louis, MO, USA). Picric acid, acetic acid, formaldehyde and paraffin wax were from Carlo Erba (Milan, IT).
5
Neutralizing activity of bispecific antibodies
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The neutralizing activity of the affinity‐matured bispecific antibodies CoV‐X2_D and CoV‐X2_F against wild‐type and Delta variant SARS‐CoV‐2 was investigated by plaque‐reduction neutralization tests following a previously reported protocol.[36 ] In brief, 50 µL of antibody, starting from a concentration of 12 µg mL−1 in a serial threefold dilution, were mixed in a flat‐bottomed tissue culture microtiter plate (COSTAR) with an equal volume of 100 median tissue culture infectious dose of infectious virus that was isolated from patients with Coronavirus disease, sequenced, titrated, and incubated at 33°C in 5% CO2. After 1 h, 3 × 104 (100 µL) Vero E6 cells (VERO C1008, Vero 76, clone 18 E6, Vero E6; ATCC CRL‐1586) were added to each well. After 3 days of incubation, the cells were stained with Gram's crystal violet solution (Merck) plus 5% formaldehyde 40% m/v (Carlo Erba S.p.A.) for 30 min. The microtiter plates were then washed in water and the wells were analyzed to evaluate the degree of cytopathic effect compared to the untreated control. Each experiment was performed in triplicate. The EC50 was determined using three‐parameter nonlinear regression (GraphPad Prism).
6
Tissue Fixation and Microscopy Analysis
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Samples of AM were fixed with 4% formaldehyde (Carlo Erba Reagents, Milan, Italy) or 3% paraformaldehyde (PFA) in 1× phosphate-buffered saline (PBS) for 2 to 3 h. Both groups of samples were then dehydrated in a series of graded increases in alcohol concentrations, embedded in paraffin, and cut on a microtome (Leica, RM 2265, Nussloch, Germany). Sections were routinely stained with hematoxylin–eosin (H&E) and Mallory trichrome staining solutions, mounted in Bio Mount, and observed under a ZEISS Axioskop 40 (Carl Zeiss, Gottingen, Germany) light microscope equipped with a Coolsnap videocamera (Photometrics, Tucson, AZ, USA).
For electron microscopy, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4, for 2 to 3 h at 4 °C. They were washed in cacodylate buffer and postfixed in 1% osmium tetroxide for 1 to 2 h at 4 °C. After dehydration in increasing alcohol gradients, samples were embedded in Spurr resin and cut at the ultramicrotome (C. Reichert-Jung Ultracut, Wien, Austria). Semithin sections were stained with 1% toluidine blue for light microscopy analysis. Observations were carried out under a ZEISS Axioskop 40 (Carl Zeiss) light microscope equipped with a Coolsnap videocamera (Photometrics).
For electron microscopy, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4, for 2 to 3 h at 4 °C. They were washed in cacodylate buffer and postfixed in 1% osmium tetroxide for 1 to 2 h at 4 °C. After dehydration in increasing alcohol gradients, samples were embedded in Spurr resin and cut at the ultramicrotome (C. Reichert-Jung Ultracut, Wien, Austria). Semithin sections were stained with 1% toluidine blue for light microscopy analysis. Observations were carried out under a ZEISS Axioskop 40 (Carl Zeiss) light microscope equipped with a Coolsnap videocamera (Photometrics).
7
Quantitative Analysis of Diuron Metabolism
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Diuron (3-(3,4-dichlorophenyl)-1,1 dimethylurea, CAS number 330-54-1, Pestanal, analytical standard), acetonitrile (anhydrous, 99.8 %), 1-chloro-2,4-dinitrobenzene (CDNB), disodium salt of reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), ultrapure reduced glutathione (GSH), hydrogen peroxide, imidazole, glutamine, and hydroxylamine were purchased from Sigma Aldrich Chemicals (France). Formaldehyde 35 %, glacial acetic acid, and butanol 100 % were purchased from Carlo Erba (France), glycerin 86 % was purchased from Roth (France), ethanol 95 % was purchased from VWR, and paraffin (paraplast®) was purchased from Leica (France). Trizol Reagent and DNAse I were purchased from Invitrogen, M-MuLV Reverse Transcriptase was purchased from BioLabs, and Syber Green Mix was purchased from Roche. Ultrapure deionized water was prepared using a Milli-Q system (Millipore, Molsheim, France). All other reagents were of analytical grade.
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