Flat bottomed 96 well microplates

Manufactured by Corning

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Flat-bottomed 96-well microplates are a type of laboratory equipment used to hold small volumes of liquids or samples in a grid-like format. These plates typically have 96 individual wells arranged in an 8x12 grid pattern, and each well has a flat bottom. The flat-bottomed design is intended to provide a stable base for the samples within the wells.

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Lab products found in correlation

Infinite m200 pro, by Tecan (2 mentions) 96 well plate, by Corning (1 mentions)

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96-well microplates (290 citations) 96-well flat-bottomed microplate (10 citations) 96 wells (10 citations) 96-well flat-bottomed polystyrene microplates (3 citations) Flat-bottomed 96-well ELISA microplates (2 citations) 96-well flat black-bottomed polystyrene microplates (2 citations)

7 protocols using flat bottomed 96 well microplates

Yeast Growth Kinetics and Cell Wall Dynamics

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S. cerevisiae strain was streaked from glycerol stocks onto YPAD agar plates and
grown for 24 h at 30 °C. Colonies were then grown in 5 mL of
YPD broth for 24 h at 30 °C with shaking in tubes. Cultures were
diluted at 1:50 and incubated in YPD broth for 3 h at 30 °C with
shaking until log-phase growth was observed. PA (final 1 mM), caspofungin
(final 1 μM), or DMSO were added from stock solutions and incubated
with shaking at 30 °C. At each time point (10 min, 3, 6, 22,
28 h) 200 μL samples were pelleted, YPD was removed, pellets
were washed twice with 200 μL of PBS and then resuspended in
200 μL of PBS. For chitin content assay, 5 μL of CFW was
added (final concentration 25 μg/mL) and incubated for 5 min
at 37 °C with shaking. For permeability assay, 1 μL of
propidium iodide (PI) was added (final concentration 5 μg/mL)
and incubated for 15 min at 37 °C with shaking. Samples were
transferred to flat-bottomed 96-well microplates (Corning) and read
by MACSQuant VYB flow cytometer. For PI assay, a 614/50 nm filter
was used. For CFW assay, a 450/50 nm filter was used.

Yona A, & Fridman M. (2024). Poacic Acid, a Plant-Derived Stilbenoid, Augments Cell Wall Chitin Production, but Its Antifungal Activity Is Hindered by This Polysaccharide and by Fungal Essential Metals. Biochemistry, 63(8), 1051-1065.

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Antifungal Susceptibility Testing Protocol

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Starter cultures were streaked
from glycerol stock onto YPAD agar plates and grown for 24 h at 30
°C. Colonies were suspended in 1 mL of PBS and diluted to 1 ×
10^–4 optical density (OD₆₀₀) in fresh
medium. Stock solutions were added to growth media, and serial double
dilutions were prepared in flat-bottomed 96-well microplates (Corning)
to enable testing of concentrations ranging from 512 to 16 μg/mL.
YPD was used as growth media. For assays of metals or polysaccharides
effect, either metal or polysaccharide in the respective concentration
(50 or 500 μg/mL of polysaccharides, and 10/100/1000 μM
of metals) was added to the YPD growth medium. Control wells with
yeast cells but no-drug (100% growth) and blank wells containing only
growth medium (0% growth) were prepared. An equal volume (100 μL)
of yeast suspensions in growth medium was added to each well except
the blank wells. Growth was determined at 35 °C by measuring
the OD₆₀₀ using a plate reader (SPARK, Tecan, equipped
with Spark-Stack) every 40 min. Each concentration was tested in triplicate,
and the results were confirmed by at least two independent sets of
experiments.

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Antifungal Susceptibility Profiling of Echinocandins

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MIC values were determined by using CLSI M27-A3 guidelines
with minor modifications. Starter cultures were streaked from glycerol
stock onto YPAD agar plates and grown for 24 h at 30 °C. Colonies
were suspended in 1 mL of PBS and diluted to 0.01 optical density
(OD₆₀₀) into fresh medium. Echinocandins and derivative
stock solutions were added to YPD broth, and serial double dilutions
of compounds in YPD were prepared in flat-bottomed 96-well microplates
(Corning) to enable testing of concentrations ranging from 64 to 0.125
μg/mL for derivatives and from 1 to 3.9 × 10^–3 μg/mL for parent drugs. Control wells with yeast cells but
no-drug and blank wells containing only YPD were prepared. An equal
volume (100 μL) of yeast suspensions in YPD broth was added
to each well with the exception of the blank wells. MIC values (Table S2) were determined after 24 h at 30 °C
by measuring the OD₆₀₀ using a plate reader (Infinite M200
PRO, Tecan). MIC values were defined as the point at which the OD₆₀₀ was reduced by ≥80% compared with the no-drug wells.
Each concentration was tested in triplicate, and results were confirmed
by two independent sets of experiments. Caspofungin was used as control
drugs.

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Antifungal Compound Screening via Growth Curves

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Growth curves were determined
using the double-dilution method in 96-well plates. Starter cultures
were streaked from glycerol stock onto YPAD agar plates and grown
for 24 h at 30 °C. Colonies were suspended in 1 mL of PBS and
diluted to 1 × 10^–3 OD₆₀₀ and then
diluted 1:100 into fresh medium. Hybrids dissolved in DMSO were added
to YPAD broth (32 μL of stock solution in 1218 μL of YPAD
broth), and serial double dilutions of hybrids in YPAD were prepared
in flat-bottomed 96-well microplates (Corning) to enable testing of
concentrations ranging from 64 to 1 μg/mL. Control wells with
yeast cells but no-drug (100% growth) and blank wells containing only
YPAD (0% growth) were prepared. An equal volume (100 μL) of
yeast suspensions in YPAD broth was added to each well with the exceptions
of the blank wells. Growth was determined at 30 °C by measuring
the OD₆₀₀ using a plate reader (Infinite M200 PRO, Tecan)
every 40 min over 48 h. Each concentration was tested in triplicate,
and results were confirmed by two independent sets of experiments.
FLC and VOR were used as control drugs.

Elias R., Basu P, & Fridman M. (2022). Fluconazole-COX Inhibitor Hybrids: A Dual-Acting Class of Antifungal Azoles. Journal of Medicinal Chemistry, 65(3), 2361-2373.

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Antimicrobial Susceptibility Assay

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The tested bacterial
strains were first grown from the frozen stock in BHI broth for 24
h at 37 °C. Volumes of 100 μL of serial 1:2 dilutions (64,
32, 16, 8, 4, 2, and 1 μg/mL) of the selected compounds in TSB
+ 1% glucose were prepared in flat-bottomed 96-well microplates (Corning).
Next, an equal volume (100 μL) of bacterial suspension in TSB
+ 1% glucose was added to each well to a final OD₆₀₀ of
0.01. Control wells with no compounds and wells without bacteria (blanks)
were also prepared. During a 24 h incubation at 37 °C, growth
kinetics were monitored by recording the optical density at a wavelength
of 600 nm (OD₆₀₀) using a Tecan plate reader. Each concentration
was tested in triplicate, and the results are shown as an average
of two independent experiments.

Kaizerman-Kane D., Hadar M., Joseph R., Logviniuk D., Zafrani Y., Fridman M, & Cohen Y. (2021). Design Guidelines for Cationic Pillar[n]arenes that Prevent Biofilm Formation by Gram-Positive Pathogens. ACS Infectious Diseases, 7(3), 579-585.

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Broth Dilution for Antifungal Susceptibility

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All strains were tested using the broth double dilution method in 96-well plates (Corning) and grown in RPMI 1640. Starter cultures were incubated for 24 h (37 °C, 5% CO₂, aerobic conditions) and then diluted 1:100 into fresh medium. Tested compounds were added to the RPMI 1640 media to form the mother liquor (32 μL stock solution in 1218 μL of RPMI 1640) at the starting concentration of 128 μg/mL. Next, 100 μL of serial double dilutions of the tested compounds in RPMI 1640 (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL) were prepared in flat-bottomed 96-well microplates (Corning). Control wells with no compounds and wells without yeast cells containing each tested concentration of the compounds (blanks) were also prepared. An equal volume (100 μL) of yeast suspensions in RPMI 1640 was added to each well for a final volume of 200 μL. Final ethanol concentrations ranged from 0.0012% to 1.3%. The final inoculum was between 5 x 10⁴ CFU/mL and 5 x 10⁵ CFU/mL.^{51 (link)} After incubation for 24 h at 37 °C in 5% CO₂, MTT (50 μL of a 1 mg/mL solution in H₂O) was added to each well followed by additional incubation at 37 °C for 2 h. Results were confirmed in two independent experiments, and each concentration was tested in triplicate.

Benhamou R.I., Bibi M., Steinbuch K.B., Engel H., Levin M., Roichman Y., Berman J, & Fridmana M. (2017). Real-Time Imaging of the Azole-Class of Antifungal Drugs in Live Candida Cells. ACS chemical biology, 12(7), 1769-1777.

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Yeast Biomass Concentration Measurement

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Yeast biomass concentration was analyzed by measuring optical density at 600 nm (OD 600 nm). Prior to the measurement the biomass was washed twice and diluted in a sterile saline solution (0.85% NaCl). OD600 measurements were done using flat-bottomed 96-well microplates (Corning; Sigma-Aldrich, USA) in Tecan Infinite M200 automatic plate reader (TECAN, Austria). All the measurements were conducted in technical duplicates of each biological replicate.

Korpys-Woźniak P, & Celińska E. (2023). Molecular background of HAC1-driven improvement in the secretion of recombinant protein in Yarrowia lipolytica based on comparative transcriptomics. Biotechnology Reports, 38, e00801.

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