After trypsinization, cell dilutions were re-suspended in media containing 10% FBS and collagen (Stemcell Technologies) on ice. Cells were then plated in U-bottomed 96-well plates (Corning) at 3000 cell/well with 0.015 mg/well collagen. After 24 h, single or multiple viable spheroids were generated. On Day 2, 4 and 6, spheroids were imaged by fluorescence microscopy (Olympus Corporation) using a 10× objective. Cell viability was measured with the CellTiter-Glo® 3D Cell Viability Assay (Promega), following the manufacturer's recommendations. Luminescence was measured at room temperature using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc).
U bottomed 96 well plates
Manufactured by Corning
Sourced in United States
The U-bottomed 96-well plates are a type of laboratory equipment used for a variety of applications, such as cell culture, biochemical assays, and drug screening. These plates feature a U-shaped well bottom, which provides a larger surface area for cell growth and facilitates the collection of samples. The 96-well format allows for high-throughput testing and efficient use of limited sample volumes.
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Lab products found in correlation
Anti cd28 mab, by BioLegend (3 mentions)
Celltiter glo 3d cell viability assay, by Promega (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Anti cd3 mab, by BioLegend (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Gamma counter, by PerkinElmer (1 mentions)
Na251cro4, by PerkinElmer (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Ix83 inverted microscope, by Olympus (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Cyan adp flow cytometer, by Beckman Coulter (1 mentions)
Trypan blue, by Biowest (1 mentions)
Mitomycin, by Merck Group (1 mentions)
2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 2h tetrazolium 5 carboxanilide xtt assay kit, by Sartorius (1 mentions)
Ficoll solution, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
10 protocols using u bottomed 96 well plates
1
3D Spheroid Cell Viability Assay
2
Cr-release Cytotoxicity Assay for CD8+ T cells
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The conventional standard [51Cr]-release assay was performed as follows. In brief, WT and LIME-/- mice were injected intraperitoneally with 106 plaque forming unit (PFU) of lymphocytic choriomeningitis virus (LCMV, Amstrong strain). Five days after immunization, CD8+ T cells were purified and mixed with gp33-41 peptide-pulsed, [51Cr]-loaded target cells (2 × 104 cells/well) in triplicate at the indicated effector/target cell ratio in 200 μl complete RPMI medium. [51Cr]-labeling of target cells were performed by incubating EL4 lymphoma cells with 200 μCi (3.7 MBq) of Na251CrO4 (Perkin Elmer, USA) for 1 h at 37°C. After 4 h of co-culture in U-bottomed 96 well plates (Corning, USA), 100 μl of supernatant was collected and the released radioactivity was measured by γ-counter (Perkin Elmer). The percentage of released radioactivity was calculated from the total radioactivity of loaded target cells per well.
3
Quantifying Biofilm Formation via Crystal Violet
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Measurement of biofilm formation via crystal violet staining was performed as described previously [41, 42 ]. In short, isolates were grown in M63 medium supplemented with 0.4 % arginine to promote biofilm formation [42 (link)]. Cultures were grown for 24 h in U-bottomed 96-well plates (Corning), at 37 °C without shaking, and OD600nm was measured. Wells were washed out with water to remove planktonic cells and biofilms were stained with crystal violet. Acetic acid was used to dissolve the stain into an aqueous solution for measurement. Measurements were made at 550 nm and normalized to the OD600nm prior to staining.
4
3D Tumor Spheroid Viability Assay
5
Evaluating Cell Viability under Hypoxia
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J-lat or ACH2 cells were resuspended in complete RPMI at a concentration of 2 × 106 cells/ml and stimulated with TNFα (100 µg/ml) or Romidepsin (20 nM) in the presence or absence of DMOG (0.5 mM) or NCS-134754 (1 µM). Cells were plated into U-bottomed 96-well plates (Corning) in a final volume of 200 μL/well and incubated at 20% O2 or 1% O2. After 24 h cells were stained for 10 min at room temperature with Live Dead Aqua to measure cell viability (Life Technologies) and fixed with 4% PFA (Santa Cruz) for 10 min at room temperature. All samples were acquired on a Cyan ADP flow cytometer (Beckman Coulter) and data analyzed using FlowJo 9.9.4 (TreeStar).
6
Immunomodulatory Effects of HUCMSCs
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The immunomodulation activity of the HUCMSCs was demonstrated with MLR. Human PBMCs from two different donors (male, 25 and 40 years old) who provided informed consent were isolated from heparinized blood by gradient centrifugation with Ficoll solution (Sigma-Aldrich) at 400 × g for 20 min at room temperature. Stimulator PBMCs were prepared by treatment with mitomycin (Sigma-Aldrich) at 25 μg/ml for 30 min at 37°C. Cell count and viability were assessed by trypan blue (Biowest) dye exclusion and then used directly in the MLR. The HUCMSCs were plated in triplicate at passage 2 into U-bottomed 96-well plates (Corning) at 10 5 cells/ ml in 100 μl of FCS-DMEM and allowed to adhere to the plate for 1 to 2 h. Human responder (10 5 PBMCs) and an equal number of stimulator PBMCs were added to the wells in 100 μl of RPMI-1640 (Invitrogen) in 10% inactivated FCS (Sigma-Aldrich). The cultures were incubated at 37°C in 5% CO 2 for 5 days, and the suspended responder PBMCs were then counted using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide (XTT) assay kit (Biological Industries). To test the role of HLA-G in MLR, 3 μg/well of HLA-Gneutralizing antibodies (87G Abs) or control antibody (20 μg/ml) (Exbio Antibodies, Praha, Czech Republic) was added on the first day of the MLR cultures.
7
Evaluating RSV-induced Immunosuppression in G-MDSC
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Granulocytic myeloid‐derived suppressor cells isolated from spleens of tumor‐bearing mice were cultured with RSV in different concentrations overnight. After incubation, G‐MDSC in different groups were collected, washed, counted again and then used for co‐culture with CD8+T cells. CD8+T cells were sorted from the splenocytes of wild‐type C57BL/6 mice with a CD8+T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. The purity of CD8+ T cells was >95%. For the T‐cell proliferation assay, CD8+T cells were incubated with CFSE (1 μmol/L, Thermo Fisher Scientific, Shanghai, China) for 20 minutes, and then equal volumes of serum‐free RPMI‐1640 medium were added to neutralize the combination. After 5 minutes, CD8+T cells were plated onto the U‐bottomed 96‐well plates (Costar) pre‐coated with anti‐CD3 (5 μg/mL; BioLegend) and anti‐CD28 mAbs (2 μg/mL; Biolegend) in triplicate and co‐cultured with different ratios of preconditioned G‐MDSC. After 2 days, cells were harvested and stained with PE‐conjugated antibodies against surface markers CD8 (BioLegend). Cell proliferation was evaluated by flow cytometry after gating on the CD8+T population.
8
Evaluating G-MDSC Suppression of T cells
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G-MDSCs were transfected with Pvt1 siRNA or the negative control. Responder cells (splenic CD4+ T cells) were cocultured with transfected G-MDSCs in U-bottomed 96-well plates (Costar, Corning, NY) in the presence of 10 μg/mL anti-CD3 mAb and 5 μg/mL anti-CD28 mAb (Biolegend, San Diego, CA) for 72 h and then pulsed with [3H]-thymidine (Pharmacia Biotech, Stockholm, Sweden, 1 μCi/well) for the last 16 h of culture. The capacity of G-MDSCs to suppress T cells was calculated according to the cpm value.
To detect the suppression function of G-MDSCs induced from bone marrow cells using IL-6 and GM-CSF treatment. 1 × 107/mL splenic CD4+ T cells were stained with the fluorescent dye CFSE (5 μM, Invitrogen) at 37 °C for 10 min and keep protected from light. 5 times the original staining volume of RPMI 1640 (Gibco, Carlsbad, CA) complete medium consisting of 10% fetal calf serum were added, pellet cells were washed twice with RPMI 1640 medium. CFSE labeled CD4+ T cells were cocultured with BM-derived G-MDSCs in round-bottomed, 96-well plates (Costar, Corning, NY) in the presence of 10 μg/mL anti-CD3 mAb and 5 μg/mL anti-CD28 mAb (Biolegend, San Diego, CA) for 72 h. CD4+ T cell proliferation was measured by CFSE dilution using FACSCalibur.
To detect the suppression function of G-MDSCs induced from bone marrow cells using IL-6 and GM-CSF treatment. 1 × 107/mL splenic CD4+ T cells were stained with the fluorescent dye CFSE (5 μM, Invitrogen) at 37 °C for 10 min and keep protected from light. 5 times the original staining volume of RPMI 1640 (Gibco, Carlsbad, CA) complete medium consisting of 10% fetal calf serum were added, pellet cells were washed twice with RPMI 1640 medium. CFSE labeled CD4+ T cells were cocultured with BM-derived G-MDSCs in round-bottomed, 96-well plates (Costar, Corning, NY) in the presence of 10 μg/mL anti-CD3 mAb and 5 μg/mL anti-CD28 mAb (Biolegend, San Diego, CA) for 72 h. CD4+ T cell proliferation was measured by CFSE dilution using FACSCalibur.
9
Maturation of DCs by LPS Induces Allogeneic CD4+ T Cell Responses
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Maturation of DCs was induced by treatment with LPS (20 ng/ml) for 24 h. In all cases, DCs were washed, counted, and replated for incubation with freshly isolated allogeneic CD4+ T cells (160,000 cells/well) in U-bottomed 96-well plates (Costar). Thus, co-cultures were always free of exogenous IL-4, GM-CSF, PGE2, or TGF-β.
10
CD4+ T Cell Proliferation Assay
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CD4+T cells were sorted from wild type C57BL/6 mice using CD4 microbeads (Miltenyi Biotec), labeled with carboxyfluorescein succinimidyl amino ester (CFSE) using the CellTraceTM CFSE Cell Proliferation kit (Invitrogen, Carlsbad, CA, USA), and were co-cultured with different ratios of 5-FU and maca polysaccharides in U-bottomed 96-well plates (Costar) in the presence of 10 μg/ml anti-CD3 mAb (Biolegend) and 5 μg/ml anti-CD28 mAb (Biolegend) for 72 h. The control group contained only CD4+T cells activated with anti-mouse CD3e and CD28 functional grades.
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