ELISA was performed in flat-bottom polystyrene plates (Greiner Bio-One GmbH, Frickenhausen, Germany) precoated with 50 μl of purified antigen diluted to 5 μg/mL in 0.05 M sodium carbonate Na2CO3 pH 9.0, overnight at 4 °C. Next, the plates were washed with PBS 0.1% Tween 20 and incubated in blocking buffer (1% Block Ace/PBS-0.1% Tween 20) at 37 °C for 1–2 h. After washing, the samples (diluted with blocking buffer to 1:100), positive controls (mouse polyclonal anti-DHCR24 sera diluted to 0.5, 1, and 2 μg/mL), and blanks (assays without sample) were added and the plates were incubated at 37 °C for 1–2 h, followed by washing. Subsequently, the secondary Abs (1:1000; peroxidase conjugate polyclonal rabbit anti-human IgG [Dako] for samples, and peroxidase conjugate polyclonal rabbit anti-Mouse IgG [Dako] for positive controls) were added and incubated at 37 °C for 1 h. Finally, the plates were washed to remove the secondary Abs, tetramethylbenzidine/H2O2-TMB (Bio-Rad, Hercules, CA, USA) was added, and the plates were incubated at 37 °C for 30 min. The reaction was stopped with 1 N of H2SO4. The absorbance values were read using a plate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. The assay was done in blind and interpreted by tree researchers. In addition, the assay was repeated for approximately 3% of the total samples, and no discrepancy was observed.
Flat bottom polystyrene plates
Manufactured by Greiner
Sourced in Germany, Austria
Flat-bottom polystyrene plates are a type of laboratory equipment used for various applications. They are made of polystyrene material and feature a flat bottom design. The primary function of these plates is to provide a stable and uniform surface for various laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using flat bottom polystyrene plates
1
Enzyme-Linked Immunosorbent Assay (ELISA) Protocol
2
Quantifying Bacterial Biofilm Biomass
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S. Enteritidis was inoculated in TSB (Tryptic Soy Broth) medium at an optical density (OD600nm) of 0.05 measured at a wavelength of 600 nm. Bacteria were then grown in 96-well flat-bottom polystyrene plates (Greiner) at 37 °C under static conditions with or without treatment. After 24 h, S. Enteritidis biofilms were washed with distilled water (dH2O) and stained for 1 h at room temperature (RT) with a 0.5% aqueous crystal violet solution. Next, the stained wells were washed 3 times with dH2O and the crystal violet-stained biofilms were then solubilized in 95% ethanol solution. Finally, relative biofilm biomasses were quantified at OD595nm using the Infinite 200 PRO microplate plate reader (Tecan). The biofilm biomasses of S. marscescens, P. aerugunosa, H. alvei, C. freundii, S. aureus, B. subtilis, S. epidermidis and E. feacalis were measured following the same staining procedure.
3
C2C12 Myoblast Viability on SG/Ch/PVA Scaffolds
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The cross-linked SG/Ch/PVA nanofiber
scaffolds were transferred
into a 96-well Flat Bottom Polystyrene plates (Greiner Bio-One, Kremsmünster,
Austria) and conditioned with 200 μL of DMEM high glucose supplemented
(10% of FBS, 1% ofl -glutamine, and 1% of antibiotics) at
37 °C and 5% CO2 for 24 h. After conditioning, the
medium was removed from the wells. Then, the C2C12 cells were trypsinized
(0.25% Trypsin-EDTA) and counted with an automated cell-counter TC10
(BioRad; California, USA) using trypan blue dye. Subsequently, myoblasts
were cultured over the conditioned SG/Ch/PVA nanofiber scaffolds at
a seeding density of 3 × 103 cells/well in 100 μL
of DMEM high glucose supplemented with 10% of FBS, 1% ofl -glutamine, and 1% of antibiotics. Cells cultured over the flat white
polystyrene wells (FPW) without the nanofiber scaffolds, with DMEM
high glucose supplemented with 10% of FBS, 1% ofl -glutamine,
and 1% of antibiotics and with or without DMSO 20% (w/w), were used
as positive and negative controls, respectively. The C2C12 cells were
incubated in a humidified atmosphere of 5% CO2 at 37 °C
for 4, 24, 48, and 72 h before performing the cell viability assay.
scaffolds were transferred
into a 96-well Flat Bottom Polystyrene plates (Greiner Bio-One, Kremsmünster,
Austria) and conditioned with 200 μL of DMEM high glucose supplemented
(10% of FBS, 1% of
37 °C and 5% CO2 for 24 h. After conditioning, the
medium was removed from the wells. Then, the C2C12 cells were trypsinized
(0.25% Trypsin-EDTA) and counted with an automated cell-counter TC10
(BioRad; California, USA) using trypan blue dye. Subsequently, myoblasts
were cultured over the conditioned SG/Ch/PVA nanofiber scaffolds at
a seeding density of 3 × 103 cells/well in 100 μL
of DMEM high glucose supplemented with 10% of FBS, 1% of
polystyrene wells (FPW) without the nanofiber scaffolds, with DMEM
high glucose supplemented with 10% of FBS, 1% of
and 1% of antibiotics and with or without DMSO 20% (w/w), were used
as positive and negative controls, respectively. The C2C12 cells were
incubated in a humidified atmosphere of 5% CO2 at 37 °C
for 4, 24, 48, and 72 h before performing the cell viability assay.
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