Drosophila Schneider S2 cells (kind gift from Prof. Jianguo He, Sun Yat-sen University, Guangzhou, China) were cultured at 27°C in Schneider's Drosophila Medium (Cat# 21720-024, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Cat# 10091148, Gibco Life Technologies, Grand Island, NY, USA). Confluent cells were seeded onto 24-well plates (Cat# 725021, NEST Biotechnology, Shanghai, China) at a density of 1 × 106 cell/mL per well in a volume of 500 μL medium. At 60–80% confluence, cells were transfected with 250 ng of pIZ-PvHMCs-EGFP-Flag plasmid or with an equal amount (250 ng) of pIZ-EGFP-Flag using the FuGENE HD Transfection Reagent (Cat# E2311, Promega, Madison, WI, USA) according to the manufacturer's instructions. At 48 h post-transfection, cells were harvested and washed three times with pre-cooled PBS before being lysed in IP Lysis Buffer (Cat# CW2334S, Pierce) containing protease inhibitor cocktail (Cat# 87785, Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were centrifuged at 20,000 g for 20 min at 4°C to collect the supernatant. Next, lysates were mixed with 5× loading buffer, boiled for 10 min before being analyzed by SDS-PAGE and Western blot as previously described (Aweya et al., 2020 (link)).
24 well plate
Manufactured by NEST Biotechnology
Sourced in China, United States
24-well plates are a common laboratory equipment used for cell culture and various assays. They provide a standardized platform with 24 individual wells, allowing for the simultaneous testing or cultivation of multiple samples or conditions. The core function of 24-well plates is to serve as a container and support structure for cell-based experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Transwell chamber, by Corning (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
8.0 m pet track etched membrane transwell, by BD (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Duo litetm luciferase assay system, by Vazyme (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Sf 900 2 sfm, by Thermo Fisher Scientific (2 mentions)
Tc 100 insect medium, by AppliChem (2 mentions)
H4000, by Engreen Biosystem (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Osteogenic induction medium, by Cyagen (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Spectramax m3 spectrophotometer, by Molecular Devices (2 mentions)
Piercetm 660 nm protein assay reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Completetm protease inhibitor cocktail, by Roche (2 mentions)
65 protocols using 24 well plate
1
Drosophila S2 Cell Transfection and Lysis
2
Cell Migration and Invasion Assay
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Transwell chambers (8.0 μm, Corning Life Science, USA) were inserted in 24-well plates (Nest, China). A 200 μL volume of serum-free medium containing 1×105 cells and 600 μL of complete medium containing 10% FBS were added to the upper and lower chambers of each well, respectively. The cells were further incubated for 24 h. The cells were fixed in 4% paraformaldehyde at room temperature for 30 min, followed by crystalline violet staining (Beyotime, China) for 30 min. The cells in the upper chamber were then washed in PBS, and were gently wiped with a cotton swab. Three fields of view were randomly selected under the microscope to observe the cells and were photographed for counting (20x magnification). For invasion experiments, Matrigel (Corning Life Science, USA) was mixed with DMEM in a ratio of 1:6 and 30 μL was added to each chamber and placed in an incubator at 37°C for 5 h. The remaining steps were performed as for Transwell experiments.
3
Transwell Assay for Cell Migration
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Cell transwell experiments were carried out using 8.0 µm PET track-etched membrane transwell (BD Falcon, Franklin Lakes, NJ, USA), with each transwell inserted into the 24-well plates (NEST, Shanghai, China) to form an upper and a lower chamber. DLD-1 and HCT116 cells were pretreated with DIM (0, 40 μM) for 48 h. To elucidate the effect of DIM on migration ability, serum-free medium (200 µL) containing 2 × 105 DLD-1 and HCT116 cells was added to the upper layer. Then, 600 µL complete medium containing 10% FBS was added into lower chambers. These chambers were incubated in the humidified incubator for 24 h. The compartment was removed, and we gently wiped off the upper cells with a cotton swab. The migrative cells in the lower layer of the chamber were fixed with 4% paraformaldehyde for 10 min. After washing with PBS twice, the cells were stained with 0.05% crystal violet for 15 min. Photographs were taken for counting under a microscope.
4
Luciferase Reporter Assay for IFN-β
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The DF-1 or GEF cells were plated in 24-well plates (NEST Biotechnology, Wuxi, China) and transiently transfected with the reporter plasmid pGL-IFN-β-Luc (0.12 μg/well) and internal control Renilla luciferase (PRL-TK, 0.06 μg/well) along with the indicated plasmids using Nulen PlusTrans™ Transfection Reagent (Nulen, Shanghai, China). According to the manufacturer’s instructions, the cells were lysed 24 hours after transfection, and luciferase activity was measured using a Dual-Luciferase Reporter Assay System kit (Promega, USA). Renilla luciferase activity was employed for normalization. All reporter assays were repeated at least three times.
5
Colorectal Cancer Cell Migration and Invasion Assay
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For the migration and invasion assay, 8.0 µm PET track-etched membrane transwell (BD Falcon, Franklin Lakes, NJ, USA), with each transwell inserted into the 24-well plates (NEST, Shanghai, China) to form an upper and a lower chamber, and Matrigel (BD) were used for estimating cell invasion. HCT116, DLD1 and RKO cells were pretreated with different indicated concentration of C.B CM for 24 h. Then C.B CM treated cells (1 × 105 (link) cells for migration, or 1.5 × 105 (link) cells for invasion) in 200 µL of serum-free media were seeded into upper chambers. RPMI 1640 or DMEM supplemented with 10% FBS was placed in the lower chamber. Migration and invasion were scored at 12 and 24 h, respectively. The migrated cells in the lower layer of the chamber were fixed with 4% paraformaldehyde for 5 min at room temperature, and stained with crystal violet for 15 min. Photographs were taken for counting under a microscope.
6
Isolation and Purification of CD133+CD34+ Cells from Umbilical Cord Blood
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hUCB samples (60–80 ml/unit) were obtained from healthy full-term births with parental informed consent at the Southwest Hospital, Third Military Medical University (Army Medical University). Fresh umbilical cord blood cells were separated within 12 h and used in subsequent experiments. Human mononuclear cells were isolated by density-gradient centrifugation with Ficoll-Paque PREMIUM 1.077 g/ml (GE Healthcare, Little Chalfont, United Kingdom). Red blood cells were removed with red cell lysate. According to the manufacturer’s instructions, CD133+ cells were isolated from human monocyte cells with a human-CD133 MicroBead Kit (130-100-830, Miltenyi Biotec) by magnetic bead separation. To assess the sorting, CD133+ cells were stained by primary PE-conjugated CD133 antibody (130-113-670, Miltenyi Biotec). The cell purity was 95%. CD133+ cells were seeded at 2 × 105 cells/well in 24-well plates (NEST, China) and then cultured in StemMACS™ HSC Expansion Media, human (130-100-463, Miltenyi Biotec) at 37°C in a humidified atmosphere containing 5% CO2 for 7 days, then PE-conjugated anti-human-CD133 (130-113-670, Miltenyi Biotec) and APC-conjugated anti-human-CD34 (130-113-176, Miltenyi Biotec) antibodies were used to obtain CD133+CD34+ cells by flow sorting.
7
Evaluating Treg and Teff Response to Oxidative Stress
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Flow cytometry sorted CD4+CD25high Tregs and CD4+CD25- Teffs were plated in 24-well plates (Wuxi Nest Biotechnology Co., Ltd, Wuxi, China), and cultured in RPMI-1640 (HyClone) containing 10% FBS (Gibco) and antibiotics for 24h. Then cells were treated with a subcytotoxic concentrations of H2O2 (100 µM) for 2 hours as a stress inducer. All cells were incubated at 37°C in a humidified 5% CO2 atmosphere incubator.
8
miR-383-5p Target Gene Validation
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The MiRanda database (https://www.microRNA.org ; accessed on 1 March 2022) was used to predict miR-383-5p-related target genes. The potential binding site between miRNA and mRNA was predicted using the RNA22 website (https://cm.jefferson.edu/rna22/Interactive/ ; accessed on 15 January 2023), based on the sequence information. According to the results, RAD51AP1 was selected as the possible target gene of miR-383-5p. Luciferase reporter plasmids (wild-type (WT) and mutant (MUT) of target sequence) were constructed by Tsingke Biotechnology Co., Ltd. (Beijing, China). Next, 293T cells were seeded into 24-well plates (NEST Biotechnology, Wuxi, China). The miR-383-5p mimic or NC was co-transfected with specific WT or mutant plasmids using the lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) when the cell density reached 70%. Then, luciferase activities were measured using the Duo-Lite TM Luciferase Assay System (Vazyme, Nanjing, China) after Aa24 h.
9
Cytotoxicity Assay of Bacterial Cultures
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VERO cells were cultivated in 24-well plates (Nest, USA) in DMEM medium (Cultilab Ltd.) containing 10% FBS, for 48 h at 37ºC, in a 5% CO2 atmosphere. The isolated strains were cultivated in LB broth for 18 h at 37ºC. An aliquot was centrifuged at 13,000 g for 2 min and the supernatant was filtered through 0.22 µm membrane filters (Millipore). Bacterial culture and supernatant filtrates aliquots were also boiled for 10 min. Fifty microliters of bacterial culture, supernatant, boiled bacterial culture or boiled supernatant were added to VERO cells, and after incubation for 18 h at 37ºC, the cells were washed three times with PBS, fixed with methanol and stained using May-Grünwald and Giemsa solutions. The results were compared with control cells. Cytotoxicity was considered positive when damaged cells were observed.
10
Streptococcus mutans Biofilm Formation
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S. mutans (ATCC 25175), the standard strain isolated from carious dentin, was achieved from the School of Stomatology, Wuhan University. After 24 h of anaerobic cultivation in BHI broth at 37 °C, the S. mutans suspension was collected. The inoculation medium of S. mutans with the concentration at 108 CFU/mL was adjusted before usage. In accordance with the same approach as described in the aforementioned sections, additional third molars were sliced to produce 60 qualified dentin discs and subjected to the EDTA etching. The discs were randomly assigned to three groups (control, CPP–ACP, and EGCG@nHAp@MSN groups; n = 20 each) with 2 h of ultraviolet disinfection on each aspect followed by storage in sterile deionized water at 37 °C (half for 1 week of storage, and the other half for 1 month of storage). Twenty discs (10 for a 1-week test, 10 for a 1-month test) from each group were transferred into 24-well plates (NEST Biotechnology, Wuxi, China). Subsequently, 1 mL of S. mutans medium supplemented with 1% (w/v) sucrose was injected into each well. After 24 h of biofilm formation under anaerobic environment at 37 °C, all discs were collected. Nonadherent bacteria were gently rinsed away by using sterile PBS, and all discs were placed in another 24-well plate.
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