Isolated Teff cells (5 × 104/well) were cultured in round bottom 96-well plates (Costar) in the presence or absence of soluble anti-CD3 mAb (0.7 μg/ml; R&D). PICL (1, 0.1, 0.01 μM), PF-04957325 (1, 0.1, 0.01 μM), alone or in combination, or vehicle control (0.1% DMSO in media) were added at 0 h (Vang et al., 2013 (link)). Proliferation of popliteal LNC in response to MOG35−55peptide with inhibitors or vehicle control was performed in round bottom 96-well plates (Costar) at a concentration of 2 × 105 cells/well. After 48 h, 2 μCi per well of [3H]thymidine (NEN) was added and cells were harvested 16 h later using a semiautomated cell harvester. [3H]thymidine incorporation was determined by scintillation counting.
96 well round bottom plate
Manufactured by Corning
Sourced in United States, United Kingdom
The 96-well round-bottom plates are a type of laboratory equipment designed for various applications in scientific research. These plates feature a circular well configuration with a rounded bottom, providing a uniform surface for various experimental procedures. The plates are typically made of high-quality materials to ensure durability and consistency during use.
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Ficoll paque plus, by GE Healthcare (11 mentions)
Ze5 cytometer, by Bio-Rad (11 mentions)
Rpmi 1640, by Thermo Fisher Scientific (10 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (6 mentions)
Human ab serum, by Gemini Bio (6 mentions)
Expifectamine cho reagent, by Thermo Fisher Scientific (6 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (5 mentions)
Facsaria, by BD (5 mentions)
Lymphoprep, by STEMCELL (5 mentions)
Pyruvate, by Thermo Fisher Scientific (5 mentions)
2 mercaptoethanol, by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Alexa fluor647 labeled goat anti human igg secondary ab, by Jackson ImmunoResearch (4 mentions)
Optipro sfm, by Thermo Fisher Scientific (4 mentions)
Celltrace violet, by Thermo Fisher Scientific (3 mentions)
Ver 10, by FlowJo (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
231 protocols using 96 well round bottom plate
1
Teff Cells Proliferation Assay
2
3D Tumor-Macrophage Co-Culture Spheroids
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Three-dimensional (3D) co-cultured multicellular tumor spheroids of tumor cells (4T1 cell line) and macrophages (RAW264.7 cell line) were developed using a liquid-overlay system according to the culture scheme illustrated in Figure 6A .30 (link) RAW264.7 cells were stained with 10 μg/mL 1,1‘-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) prior to spheroid formation. 4T1 cells (1×104 cells) and DiI-labeled RAW264.7 cells (0.5×104 cells) were mixed with DMEM containing 10% FBS and 2.5% Matrigel, seeded in a 96-well round bottom plate (Corning, Corning, NY, USA), and centrifuged at 1000× g for 10 min. After 3 days, separate groups of co-cultured tumor spheroids were incubated with Lipo, PEG-Lipo, and Man-PEG-Lipo for 8 h. At the end of incubation, the co-cultured tumor spheroids were washed three times with cold PBS, and fixed with 4% PFA for 1 h at room temperature. The fluorescence intensity of different depths of the co-cultured tumor spheroids was determined using CLSM.
3
Antibiotic Susceptibility of A. baumannii
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A. baumannii strains 98-37-09, AB5075, and corresponding mutants of interest were propagated overnight in LB broth at 37 °C, diluted into fresh medium (1:100), and grown to OD600nm 0.4–0.5 at 37 °C with aeration. A total of approximately 1 × 105 cells were transferred to individual wells of a 96-well round bottom plate (Corning Life Sciences, Corning, NY, USA) containing 100 μL of LB (negative control) or 100% human serum (AEMR inducing condition) supplemented with 0, 0.125, 0.25, 0.5, 1, 2, or 4.0 µg mL−1 minocycline, levofloxacin, ofloxacin, or with 0, 25, 50, 100, or 200 µg mL−1 DMACA-labeled ofloxacin (described below) and incubated at 37 °C for 48 h. To quantify the antimicrobial effects of each antibiotic, cells were serially diluted in 0.8% sodium chloride and plated on LB agar to enumerate the viable number of CFU mL−1 for each strain during growth in LB and serum supplemented with various concentrations of the indicated antibiotic. All experiments were performed at least 3 times, and results were averaged and presented with ±standard deviation.
4
Quantitative Analysis of RBC Agglutination
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For whole blood smears, fresh human whole blood was incubated with antibody overnight at 37°C, 5% CO2. The following day, a drop of blood was smeared onto a glass slide using a glass ‘spreader’ slide. Smears were air-dried and imaged with an EVOS light microscope using the 40X objective. Images were analyzed qualitatively for evidence of RBC agglutination. For flow-based quantitative analyses, purified human RBCs were incubated with antibody for 0.5 hour at 37°C in a 96-well round-bottom plate (Corning), followed by incubation at 4°C overnight and flow cytometry-based assessment of RBC doublets.
5
Isolation and Culture of Enteric Neurospheres
6
Allogeneic CD34+ cell-derived dendritic cell-mediated T-cell proliferation
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After isolation of CD34 to generate CB-DCs, the resultant CD34− fraction was enriched for T lymphocytes using anti-CD3 magnetic microbeads (Miltenyi). For mixed leukocyte reaction (MLR), allogeneic lymphocytes (1 × 106/mL) were then labeled with cell trace violet (5 µM; Invitrogen) and cocultured with CB-DCs (2 × 105/mL) in a 96-well round-bottom plate (Corning) at a stimulator:responder ratio of 1:5. Unstimulated cell-trace-violet-labeled cells served as negative control. After 4 days, cells were stained with CD3, CD4, and CD8 and analyzed using a FACScanto (BD). T-cell proliferation analysis was performed using the proliferation tool in flowjo (Tree Star, Inc.), providing the division index.
7
Cytotoxicity Assay for NK Cell Function
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The cytotoxicity test used cryopreserved PBMC samples from patients. Two million freshly thawed PBMCs were washed twice and transferred to a 96-well round-bottom plate (Corning) with RPMI 1640 supplemented with 10% fetal calf serum and 1,000 IU/ml interleukin 2 (IL-2; Beijing Double-Crane Pharmaceutical Co., Ltd.) for 10–14 h. PBMCs were cultured in preparation for both spontaneous and IL-2-stimulated NK cytotoxicity assays. The cytotoxicity and cytokine secretion of the NK cells was determined by CD107a and IFN-γ expression against the target K562 cells at an effector-to-target ratio of 5:1 for 4 h, with GolgiStop (0.7 µl/ml, BD Biosciences) added after 1 h to trap proteins in the cytoplasm. After fixation and permeabilization of PBMCs using a Pharmingen Intracellular Staining kit (BD Pharmingen, San Diego, CA, USA) for 40 min at 4°C, we washed it twice and added the IFN-γ antibody.
8
T Cell Cytotoxicity Assay Protocol
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Target cells were washed and suspended at 1 × 106 cells/mL in R/10 (RPMI1640 with 10% FBS). One hundred thousand target cells of each type were added to each of 2 wells of a 96-well round bottom plate (Corning). Effector T cell cultures were washed and suspended at 1 × 106 cells/mL in R/10. One hundred thousand effector T cells were combined with target cells in the indicated wells of the 96-well plate. Additionally, wells containing only T cells were prepared. The plates were incubated at 37°C for 18 to 24 h. After incubation, the supernatant was harvested and subjected to ELISA using standard methods (Pierce, Rockford, IL).
9
Antimalarial Potency Assay of Plasmodium falciparum
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Plasmodium falciparum blood stage 3D7 (wild type) parasites were grown (as previously described [25 (link)]) in complete RPMI 1640 medium (RPMI 1640 medium with 2 mMl -glutamine, 25 mM HEPES, 2 g/l NaHCO3, 27.2 mg/l hypoxanthine and 0.5% Albumax II, pH 7.4) using O+ human red blood cells at 37 °C in an incubator with 5% CO2, 5% O2 and 90% N2. Sorbitol treatment was used to synchronize the parasites [26 (link)]: parasites were treated with 5% sorbitol for 20 min at room temperature to lyse trophozoite and schizont stage parasites. Dead parasites were removed by two washes with incomplete RPMI medium (RPMI 1640 medium with 2 mM l -glutamine, 25 mM HEPES, pH 7.4). Following sorbitol treatment parasites were transferred to complete RPMI 1640 medium.
To determine the viability of parasites cultures that had reached the trophozoite stage, they were diluted to 0.3% parasitaemia and 2% haematocrit and treated with increasing concentrations of NCL-00016066 in a 96-well round bottom plate (Corning) and incubated for 72 h in an incubator with 5% CO2, 5% O2 and 90% N2. Several wells were treated with either 10% DMSO or 20 µM chloroquine as controls. They were then placed in a −80 °C freezer prior to lactate dehydrogenase (LDH) and SYBR Green I assays.
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Plasmodium falciparum blood stage 3D7 (wild type) parasites were grown (as previously described [25 (link)]) in complete RPMI 1640 medium (RPMI 1640 medium with 2 mM
To determine the viability of parasites cultures that had reached the trophozoite stage, they were diluted to 0.3% parasitaemia and 2% haematocrit and treated with increasing concentrations of NCL-00016066 in a 96-well round bottom plate (Corning) and incubated for 72 h in an incubator with 5% CO2, 5% O2 and 90% N2. Several wells were treated with either 10% DMSO or 20 µM chloroquine as controls. They were then placed in a −80 °C freezer prior to lactate dehydrogenase (LDH) and SYBR Green I assays.
10
Phage-Assisted Non-Continuous Evolution (PhaNGS) Library Construction
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The PhaNGS input pool was constructed as follows: 5 L of each clone was transferred to respective wells of a 96-well round-bottom plate (Corning). Then, 100 L log phase XL-1 blue cells (Agilent, OD600 = 0.6–0.7) were added to each well before the plate was covered in a gas-permeable film (Diversified Biotech) and placed at 37 °C for 20 min. We then transferred 100 L of infected cells to respective wells of a 96-well deep-well plate (Corning Axygen) containing 400 L per well 2xYT broth with 100 g/mL carbenicillin and 1010 cfu/mL KO7 helper phage (NEB).
The deep-well plate was covered in a gas-permeable film and shaken at 1000 rpm and 37 °C for 18–24 h in an Infors HT shaker. Plates were spun down at 4,000 g for 15 min at room temperature, and the supernatant was consolidated into 50 mL tubes before adding 0.02% sodium azide and storing at 4 °C. This method leads to approximately equal quantities of each clone from a propagated supernatant (roughly 1011 cfu/mL total).
The deep-well plate was covered in a gas-permeable film and shaken at 1000 rpm and 37 °C for 18–24 h in an Infors HT shaker. Plates were spun down at 4,000 g for 15 min at room temperature, and the supernatant was consolidated into 50 mL tubes before adding 0.02% sodium azide and storing at 4 °C. This method leads to approximately equal quantities of each clone from a propagated supernatant (roughly 1011 cfu/mL total).
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