The CMRP 4489 strain was evaluated regarding floating pellicle biofilm formation in 24-well plates. An inoculum was prepared from a culture grown overnight at 28 °C in LB medium (Neogen Corporation, USA), and the suspension was adjusted according to the 0.5 McFarland scale, rendering approximately 1.5 × 108 colony-forming units/mL (CFU/mL). Afterward, 10 µL of the inoculum were added to a 24-well plate (Sarstedt, USA) containing 2 mL of LB medium, which was incubated at 28 °C for 24 h. For the swarm expansion assays, the CMRP 4489 inoculum was prepared as described above. Afterward, 90 mm-diameter Petri dishes containing LB medium with 0.7% agar (Neogen Corporation, USA) were dried in a biological safety chamber (Filterflux, Brazil) for 30 min and inoculated centrally with 10 µL of the inoculum, dried for another 10 min, and incubated at 28 °C for 24 h.
24 well plate
Manufactured by Sarstedt
Sourced in Germany, United States, Canada, Switzerland, Austria
The 24-well plates are a type of laboratory equipment used for various cell culture applications. They provide a standardized format with 24 individual wells, allowing for the simultaneous cultivation and analysis of multiple cell samples or experimental conditions.
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Lab products found in correlation
Lsr fortessa cytometer, by BD (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Turbofect, by Thermo Fisher Scientific (4 mentions)
96 well plate, by Sarstedt (3 mentions)
Fetal calf serum (fcs), by GE Healthcare (3 mentions)
Rpmi 1640, by Merck Group (3 mentions)
Gentamicin, by Merck Group (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (2 mentions)
1 ml pipette tip, by Sarstedt (2 mentions)
Glomax discover, by Promega (2 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Granulocyte macrophage colony stimulating factor gm csf, by Thermo Fisher Scientific (2 mentions)
143 protocols using 24 well plate
1
Evaluation of Biofilm Formation and Swarming Motility in CMRP 4489 Strain
2
In Vitro Anthelmintic Screening
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Five weeks to six weeks post-infection (p.i.), the worms were collected directly from the hamster’s intestines. In a 24-well plate (Sarstedt, Nümbrecht, Germany), 3 to 4 adult worms per well were incubated in 2.5 ml drug solution with 4 different concentrations ranging from 0.005 µM to 0.5 µM for A. ceylanicum and with concentrations of 0.01 µM, 0.1 µM and 1 µM for N. americanus.
3
Evaluating EBV Peptide Immunogenicity
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The immunogenic potential of EBV-derived peptides was evaluated by functional immunoassays. PBMCs were isolated from blood of healthy HLA-A*03:01+EBV+ donors by density gradient centrifugation. Freshly isolated PBMCs were rested overnight (at 37°C, 5% CO2) at a density of 1 × 107 cells/well (24-well-plate, Sarstedt) prior to short- (overnight) and long-term (7 days) in vitro stimulation. The known HLA-A*03:01-restricted EBV-derived peptide RLRAEAQVK (A*03_EBNA3ARLRA, ProImmune [17 (link), 47 (link)]) and the peptide pool PepTivator EBV Consensus (EBV_Consensus, Miltenyi Biotec) have consistently been used as referential stimulating antigens.
On day 1, cells at a density of 5 × 106 cells/well (24-well-plate, at 37°C, 5% CO2) were stimulated with one of the eleven EBV-specific peptides (10 μg/ml) or with EBV_Consensus+4PMIX (Table1 ) consisting of a combination of EBV_Consensus (1μg per peptide/ml) and the four highly immunodominant peptides (10 μg per peptide/ml). Cells were expanded in TexMACS-medium (Miltenyi Biotec) supplemented with 0.5 μl/ml interleukin (IL)-2 and 1 μl/ml IL-7 (both PeproTech) over 7 days.
On day 1, cells at a density of 5 × 106 cells/well (24-well-plate, at 37°C, 5% CO2) were stimulated with one of the eleven EBV-specific peptides (10 μg/ml) or with EBV_Consensus+4PMIX (Table
4
Evaluating Composite Material Cytotoxicity
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For the extraction of composites, the composites were immersed in 1.5 mL culture medium in a 24-well plate (Sarstedt, Nümbrecht, Germany) at 34 °C and 5% CO2. After 24 h incubation, the extracts were collected. On the previous day, hFOB 1.19 cells were seeded at a density of 5 × 104/cm in 24-well plates. After 24 h incubation, the medium was replaced with 1 mL of composite extract or fresh medium, and the plates were incubated for a further 48 h at 34 °C and 5% CO2. Each plate contained triplicates of untreated cells, and cells treated with chitosan (CH), chitosan-bioglass (CHB), chitosan-bioglass-peptide (CHBp) extracts, positive control, and blank wells. After the incubation, cell viability and cytotoxicity were measured. The experiment was repeated twice.
5
Smooth Muscle Cell Migration Assay
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A10 rat smooth muscle cells were seeded into a 24-well plate (Sarstedt) at 36,000 cells/cm2 and incubated overnight in standard growth media. Following incubation, scratches were made with a p20 pipette tip vertically through the well. Media within each well was removed, and the cells were washed with 1 mL pre-warmed PBS. The PBS was replaced with complete media supplemented with 2% (v/v) vehicle control (PBS) or 2% (v/v) whole secretome. Migration was assessed using the Nikon TiE imaging system where images were gathered every 30 min over a 16-h period. Analysis was performed on images taken at 0 h compared to those gathered at 6 h (where a ~ 50% gap closure was observed). Gap area at 6 h was quantified and presented as a percentage closure relative to that at 0 h.
6
Quantification of Bacterial Pigments
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The bacterial culture grown overnight was diluted to 1 × 105 CFU/mL in a TSB medium, transferred to a 24-well plate (Sarstedt, Nümbrecht, Germany), and incubated at 37 °C for 48 h. After incubation, each bacterial suspension was collected in an Eppendorf tube and centrifuged at 10,000 RPM. The obtained supernatants were transferred to a transparent 96-well plate (for pyocyanin measurement) and a black 96-well plate (for pyoverdine measurement). The absorbance for pyocyanin was measured using a Varioskan Lux microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) at λ = 686 nm, and the fluorescence for pyoverdine at λ = 395 nm (excitation) and λ = 460 nm (emission). Five repetitions were made for each experiment option [21 (link)]. For the statistical analysis, the one-way ANOVA (the Tukey test) for independent groups was used (p-values < 0.001 were regarded as significant).
7
Seeding RTL-W1 Cells on Thermanox Coverslips
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RTL-W1 cells were seeded onto sterilized (EtOH + 1 h UV) Thermanox™ coverslips (Ø 13 mm) placed in the wells of a 24-well plate (Sarstedt). The cell density at the time of seeding was 12 × 104 cells/well, or ~ 6.6 × 104 cells/cm2. The volume of culture medium (L-15 containing 5% FBS) per well was 1 ml.
8
Culturing RTL-W1 cells on poly-L-lysine
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RTL-W1 cells were seeded onto poly-L-lysine-coated glass coverslips (Ø 13 mm) placed in the wells of a 24-well plate (Sarstedt). The cell density at the time of seeding was 10 × 104 cells/well, or ~ 5.5 × 104 cells/cm2. The volume of culture medium (L-15 containing 5% FBS) per well was 1 ml.
9
Scratch Wound Healing Assay
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A549/NF-κB-luc cells were seeded at 1 × 105 cells/cm2 in a 24-well plate (Sarstedt) and left to reach confluence for 48 h. Single scratch wounds were generated with a 1-mL pipette tip (Sarstedt). The cells were washed with PBS, and their respective treatments were added. Ten per cent serum was also added. Cell treatments were MEM-α medium, naive FBS- or XF-MSC CM, or pre-activated FBS- or XF- MSC CM +/−, a KGF neutralization antibody (0.5 μg/mL) (R&D Systems). Wound restitution was assessed over 48 h using light microscopy imaging.
10
Fabrication of FN-silk Protein Foams
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FN-silk protein (3 mg/mL) were placed as a defined drop (15–40 µL) spread out to a diameter of 1 cm in the middle of a well of a non-treated, hydrophobic 24-well plate (Sarstedt). Air was quickly pipetted into the drop 20–30 times. Cells (0.5–2 × 106/mL) suspended in the appropriate culture media (containing 25 mM Hepes, without serum), were added dropwise (10–20 µL) either before or after introduction of air bubbles. The foams were kept 15–60 minutes in the cell incubator before covered with complete cell culture medium. For foams integrated with hESC, 15 µl of FN-silk was used for 50 000 hESCs (15 000 cells/µl). Cells were integrated in the presence of 10 µg/ml Rock inhibitor Y27632 (VWR) and silk was stabilized for 15 min at 37 °C in 5% CO2. After stabilization, 0.7 ml NutriStem supplemented with 10 µg/ml Rock inhibitor Y27632 was added. Rock inhibitor was omitted in the medium from 24 hours after seeding. Larger foams were prepared from 1.5 mL of FN-silk (3 mg/mL) subjected to 5–10 sec of whipping using a small battery operated hand whisker (Rubicson).
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