Prominin-1+ cells were selected from dissociated IC using a magnetic-activated cell sorting (MACS) system with anti-mouse prominin-1 magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol. To generate primary neurospheres, MACS-selected cells were cultured on uncoated plates at clonal density (1–2 cell/mm2) in Neurobasal medium (Life Technologies, CA, USA) containing MACS NeuroBrew-21 without vitamin A (Miltenyi Biotec) supplemented with epidermal growth factor (EGF) (20 ng/ml; Miltenyi Biotec), fibroblast growth factor-2 (FGF2) (20 ng/ml; Miltenyi Biotec), and penicillin-streptomycin-glutamine (Wako, Tokyo, Japan). The number of neurospheres was counted by microscopy following 6 days of culture at 37 °C under a 5% CO2 atmosphere. To generate secondary neurospheres, primary neurospheres were dissociated with Accutase (Life Technologies, Carlsbad CA, USA) and then seeded on new plates. For clonal cell analysis, prominin-1+ cells were seeded at one per well by the limiting dilution method or into 96-well plates (Sumitomo Bakelite, Tokyo, Japan) by a fluorescence-activated cell sorter (FACSAria I, BD). Clusters of cells > 50 μm in diameter were counted as neurospheres.
96 well plate
Manufactured by Sumitomo Bakelite
Sourced in Japan
96-well plates are a commonly used laboratory equipment item designed to hold small volumes of samples or reagents. They typically feature a grid of 96 individual wells, each with a standard volume capacity, allowing for multiple samples to be processed simultaneously. The core function of 96-well plates is to provide a standardized and organized platform for a variety of laboratory procedures and assays.
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Lab products found in correlation
Tmb one component hrp microwell substrate, by Fortis Life Sciences (2 mentions)
Tmb one component substrate, by Fortis Life Sciences (2 mentions)
Anti chicken igy peroxidase rabbit antibody, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin glutamine (psg), by Fujifilm (1 mentions)
Epidermal growth factor (egf), by Miltenyi Biotec (1 mentions)
Facsaria 1, by BD (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Macs neurobrew 21 without vitamin a, by Miltenyi Biotec (1 mentions)
Fibroblast growth factor 2 (fgf2), by Miltenyi Biotec (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Porcine skin gelatin, by Merck Group (1 mentions)
White 96 well plate, by Thermo Fisher Scientific (1 mentions)
Envisionhts multilabel reader, by PerkinElmer (1 mentions)
Celltiter glo luminescent cell viability kit, by Promega (1 mentions)
Crl 2014, by American Type Culture Collection (1 mentions)
9 protocols using 96 well plate
1
Isolation and Expansion of Neural Stem Cells
2
ELISA-based Antibody Titer Determination
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Antibody titers of each immune plasma sample were determined via enzyme-linked immunosorbent assay (ELISA). Recombinant CP-PDs (100 ng/well) were coated on wells of 96-well plates (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) and incubated in carbon-bicarbonate buffer (pH 9.8) at 4°C overnight. Thereafter, each well was blocked with PBST-containing 1% bovine serum albumin at 37°C for 2 h. After blocking, 8,000-, 16,000-, and 32,000-fold PBS-diluted immune plasma was added to each well and incubated at 25°C for 30 min. Then, wells were washed five times with PBST and incubated at 37°C with anti-chicken IgY[IgG](H+L)-HRP, Goat (Bethyl Laboratories, Inc., Montgomery, TX, USA) for 1 h. 3, 3’, 5, 5’-tetramethylbenzidine (TMB) One Component HRP Microwell Substrate (Bethyl Laboratories, Inc.) was added to each well and incubated at 37°C for 15 min. After adding 100 μL of 0.18 M H2SO4 to each well, sample absorbance was measured at 450 nm. The plasma from control chickens was diluted 2,000-fold and its absorbance was measured as previously described. The cutoff value was set at OD450 = 0.18, and the antibody titer was indicated as the maximum dilution rate.
3
Antibody Titre Determination by ELISA
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Antibody titres in the immune plasmas were determined by ELISA. The purified Dg-Ctr1-N-his was coated on the wells of 96-well plates (Sumitomo Bakelite Co., Ltd, Tokyo, Japan) (100 ng well−1) for 16 h with carbon-bicarbonate buffer. After washing each well three times with PBS, PBS containing 0.05% Tween-20 (PBS-T) with 1% BSA was added and incubated at 37°C for 2 h. Wells were then washed five times with PBS-T, and immune plasmas diluted 2000×, 4000×,8000×, 16 000×, 32 000× and 64 000× with PBS were added. After 1 h of incubation at room temperature, the wells were washed five times with PBS-T and incubated with anti-chicken IgY peroxidase rabbit antibody (Sigma-Aldrich) for 1 h at room temperature. Finally, the wells were washed five times with PBS-T and reacted with the TMB one-component substrate (Bethyl Laboratories, Montgomery, TX, USA) for 20 min at room temperature in the dark. The reaction was quenched with 0.18 m H2SO4 and the absorbance was measured at 450 nm. The assay was performed in duplicate.
4
PEDV Antibody Titer Determination
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Confluent monolayers of Vero cells grown in 96-well plates (Sumitomo Bakelite, Tokyo, Japan) were inoculated with 102.5 TCID50 OKN-1/JPN/2013 in 100 μL, and incubated at 37 °C in a humidified atmosphere with 5% CO2. Inocula were removed after 18 to 24 h. Subsequently, cells were washed with PBS once, and fixed with cold 70% acetone (FUJIFILM Wako Pure Chemical, Osaka, Japan) for 10 min. Plates were then air dried, sealed and stored at –20 °C until probed with 50 μL sera serially diluted two-fold from 1:20 to 1:1280 in PBS. Sera were removed after 1 h at 37 °C, and plates were washed 5 times with PBS. Wells were then reacted for 1h at 37 °C with 50 μL/well of 1:50 goat anti-swine IgG (H + L) conjugated to fluorescein isothiocyanate (Jackson ImmunoResearch, West Grove, PA, USA), washed five times with PBS, an examined for PEDV-specific cytoplasmic staining under a fluorescent microscope. Antibody titers are reported as the reciprocal of the highest dilution that produced clear, specific cytoplasmic staining.
5
Antibody Titration via ELISA
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Antibody titres in the immune plasmas were determined through enzyme-linked immunosorbent assay (ELISA ). The recombinant Deg-CPR-2 was coated on the wells of 96-well plates (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) (100 ng/well) at 4°C overnight with carbon-bicarbonate buffer (pH 9.8). After removing the recombinant protein, PBST-containing 1 % bovine serum albumin was added into each well. Next, the plates were incubated at 37°C for 2 h. After blocking, immune plasma diluted 8,000-, 16,000-, and 32,000-fold was added into each well; subsequently, the plates were incubated at 25°C for 30 min. After incubation, the wells were washed 5 times with PBST and incubated at 37°C with anti-chicken IgY[IgG](H+L)-HRP, Goat (Bethyl laboratories, Montgomery, TX) for 1 h. As a substrate, TMB One Component HRP Microwell Substrate (Bethyl Laboratories, Inc.) was added into each well, followed by incubation at 37°C for 15 min. After adding 100 μL of 0.18 M H2SO4 into each well, the sample absorbance was measured at 450 nm. Plasma from control chickens was diluted 2,000-fold, with its absorbance being measured as described. The cut-off value was set at optical density (OD )450 = 0.13; further, the antibody titre was indicated as the maximum dilution rate.
6
Culturing miPS-LLCcm Cells on Gelatin
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Cells of miPS-LLCcm derived from iPS-MEF-Ng-20D-17 provided by the Riken BioResource Research Center through the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology/Japan Agency for Medical Research and Development, Japan [12 (link)], were cultured in a medium containing Lewis lung cancer (LLC) cell culture-conditioned medium (cm) mixed with Dulbecco’s Modified Eagle Medium (DMEM) high glucose with 15% fetal bovine serum (FBS), 1× non-essential amino acids (NEA), 1% penicillin/streptomycin (P/S), 1× L-glutamine, and 100 μM of 2-mercaptoethanol in a ratio of 1:1 on 96-well plates (Sumitomo Bakelite, Tokyo, Japan) precoated with 0.1% porcine-skin gelatin (MilliporeSigma, St. Louis, MO, USA) following the procedure described in a previous report [3 (link)]. We grew LLC cells in DMEM high glucose supplemented with 10% FBS, 1× NEA, and 1% P/S. To obtain the cm, we maintained confluent grown cells in the medium by changing 10% FBS to 5% for 1 day; subsequently, we filtrated the cm. We sustained the cells at 37 °C in a 5% CO2 incubator (PHC, Gunma, Japan). The culture reagents were purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.
7
Enzyme-Linked Immunosorbent Assay for Antibody Titers
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Antibody titers in plasma samples were determined by ELISA. The purified Dg-Cys-his was coated onto the wells of 96-well plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) (100 ng/well) for 16 h in a carbon-bicarbonate buffer. After washing each well three times with PBS, PBS containing 0.05% Tween 20 (PBS-T) and 1% BSA was added and incubated at 37 °C for 2 h. The wells were then washed five times with PBS-T, and plasma samples diluted at 2000×, 4000× 8000×, and 16,000× with PBS were added. After incubation for 1 h at 25 °C, the wells were washed five times with PBS-T and incubated with anti-chicken IgY peroxidase rabbit antibodies (Sigma-Aldrich, A9046) diluted at 5000× with PBS-T for 1 h at 25 °C. Finally, the wells were washed five times with PBS-T and allowed to react with the TMB one-component substrate (Bethyl Laboratories, Montgomery, TX, USA) for 20 min at 25 °C, in the dark. The reaction was quenched with 0.18 M H2SO4, and the absorbance was measured at 450 nm. The assay was performed in duplicate.
8
Evaluating Tie2 Receptor Activation
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Human Tie2-expressing Ba/F3 cells were suspended in RPMI1640 medium (Thermo Fisher Scientific) supplemented with 0.05% FBS at 2 × 105 cells/mL and 80 μL was added to each well of a 96-well plate (Sumitomo Bakelite). Thereafter, 20 μL of the purified antibody or Ang 1 diluted with phosphate buffer saline (PBS) was added. After culturing for 72 h in a CO2 incubator set to 37 °C, 50 μL of the cell suspension was transferred to a white 96-well plate (Thermo Fisher Scientific). To measure intracellular ATP using CellTiter-Glo Luminescent Cell Viability Kit (Promega), 50 μL of CellTiter-Glo reagent was added into each well. The microplate was shaken and incubated in the dark. Luminescence was measured using EnVisionHTS Multilabel Reader (PerkinElmer).
9
HGF Cell Viability and ROS Assays
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The HGF cell line, CRL-2014®, was obtained from ATCC (Manassas, VA, USA). HGFs were seeded at 4,800 cells/cm2 and cultured in DMEM supplemented with 10% FBS for 5 days and reached sub-confluency. HGFs were cultured using a 96-well plate (SUMITOMO BAKELITE, Tokyo, Japan) for 5 days and used for cell viability assays. In ROS assays, HGFs were cultured using a 96-well black plate (Nunclon™, Thermo Scientific™, Waltham, MA, USA) for 5 days, and then treated for ROS assays. In other experiments, the sub-confluent HGFs were pre-treated with 2.5–15 μM 6-shogaol for 1 h and cultured with 500 μg/mL of AGEs or BSA for 0.5–48 h and used for a western blotting analysis and enzyme-linked immunosorbent assays (ELISAs).
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