For chondrogenic differentiation, 100,000 cells were added to the wells of U-bottom 96-well plates (Cat# 10861-564, VWR, Radnor, PA) following treatment with Anti-Adherence Rinsing Solution (StemCell Technologies cat# 07010). The plates were centrifuged at 100×g for 3 min for pellet formation.
U bottom 96 well plates
Manufactured by Avantor
Sourced in United States
U-bottom 96-well plates are a type of microplate used in various laboratory applications. They feature a rounded, U-shaped bottom on each well, which can be advantageous for certain experimental setups or analyses.
Automatically generated - may contain errors
Lab products found in correlation
Anti adherence rinsing solution, by STEMCELL (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axiocam icc1, by Zeiss (1 mentions)
Fc blocking antibody 2.4 g2, by BD (1 mentions)
F4 80 af700, by BioLegend (1 mentions)
Cd11c bv785, by BioLegend (1 mentions)
Cd8 percpcy5.5 53 6 7, by BioLegend (1 mentions)
Facs fortessa, by BD (1 mentions)
Cd11b pe cy7, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Zombie yellow, by BioLegend (1 mentions)
7 aad, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
4 protocols using u bottom 96 well plates
1
Chondrogenic Differentiation of Cell Pellets
2
Evaluating Cancer Spheroid Growth
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Human ovarian and breast cancer cells (parental and resistant) were seeded into U bottom 96-well plates (VWR, Radnor, Pennsylvania, United States) coated with 0.8% SeaKem LE Agarose at a cell density 0.5 × 105 cells/mL (HOC, HOC/ADR) and 1 × 105 cell/mL (MCF-7, MCF-7/PAX) in 100 µL of medium with appropriate cytostatic agent. After 24 h, the plates were enriched with 100 µL of medium with tested compounds K3 , K4 , K7 at the final concentration of 1 µM, except for control cells (100 µL medium with 1% V/V DMSO). Spheroids were recorded 0; 24; 48 and 72 h after exposure to tested compounds by using the light microscope Axio Vert. A1 (Zeiss, Jena, Germany) with photo documentation equipment, Axiocam ICC 1 and Axio Vision 4.8 software (Zeiss), and the spheroid areas were determined by using ImageJ (National Institute of Health, Bethesda, Maryland, United States). Subsequently, a spheroid growth was calculated according to equation: where SAS represents the area of spheroids treated with the compound; SAC is the control spheroid area; and the exposure time (t = 0; 24; 48; 72 h).
3
Multiparametric Phenotyping of Splenocytes
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Splenocytes were cultured in U-bottom 96-well plates (VWR) at 1 × 106 cells/200 μl of supplemented RPMI medium and incubated as described for antigen processing assays. Thereafter, cells were washed and incubated for 5 min at 4 °C with Fc blocking antibody (2.4G2) (BD Pharmingen) before stained for 20 min in the dark at 4 °C with saturating concentrations of surface-targeted antibodies and viability markers. Cells were fixed with Cytofix/Cytoperm kit (BD Biosciences), measured by FACS Fortessa (BD Biosciences) and analysed by FlowJo software (Tree Star, Ashland, OR). Dead cells were excluded using Zombie Yellow (Biolegend). Antibodies used as follows: CD3-APC (145-2c11, Biolegend), F4/80-AF700 (cat. MCA497A700, BioRad), LY6G/6C-APC-Cy7 (RB6-8C5, BD), NK1.1-BV421 (PK136, Biolegend), MHCII-BV711 (M5/114, BD), CD11c-BV785 (N418, Biolegend), CD11b-PE-Cy7 (M1/70, BD), CD45R/B220-V500 (RA3-6B2, BD), CD103-PE (2E7, Invitrogen), CD8-Percp-Cy5.5 (53–6.7, Biolegend) and CD317-BV650 (927, Biolegend).
4
Profiling T-Cell Responses to CPC and EV Immunomodulation
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Tailored mixed lymphocyte reactions (MLR) were performed using PBMC from different donors.
Briefly, responding PBMC (1x10 5 cells) were labeled with 2.5 µM carboxyfluorescein succinimidyl ester (CFSE, Thermofisher Scientific) and then co-cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco Life Technologies) in U-bottom 96-well plates (VWR) with human leukocyte antigen mismatched (HLA) mitomycin-C-treated (Sigma) stimulatory PBMC (1x10 5 cells), mitomycin-C-treated CPC (at CPC:PBMC ratios of 1:5 and 1:10), or increasing doses of EV (EV1: 5.5x10 8 , EV2: 5.5x10 9 or EV3: 1.7x10 10 particles) or PBS as a control. After 6 days of co-culture, cells were harvested and stained with anti-CD3-PE/Cy7, anti-CD4-APC, anti-CD8-APC-H7 antibodies and 7AAD (BD Biosciences). Proliferation and cell death of T-cell subsets were monitored by FACS.
In assays determining the immunomodulatory effects of CPC and EV-CPC, HLA-mismatched CFSE-labeled PBMC (1x10 5 cells) were stimulated with 1 μg/mL phytohemagglutinin (Sigma-Aldrich) in the presence or absence of mitomycin-C-treated CPC (at CPC: PBMC ratios of 1:5 and 1:10) or with increasing concentrations of EV (EV1: 5.5x10 8 , EV2:5.5x10 9 or EV3: 1.7x10 10 particles) for 5 days. At the end of the co-culture, the proliferation and death rate of the T cells were monitored by FACS.
Briefly, responding PBMC (1x10 5 cells) were labeled with 2.5 µM carboxyfluorescein succinimidyl ester (CFSE, Thermofisher Scientific) and then co-cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco Life Technologies) in U-bottom 96-well plates (VWR) with human leukocyte antigen mismatched (HLA) mitomycin-C-treated (Sigma) stimulatory PBMC (1x10 5 cells), mitomycin-C-treated CPC (at CPC:PBMC ratios of 1:5 and 1:10), or increasing doses of EV (EV1: 5.5x10 8 , EV2: 5.5x10 9 or EV3: 1.7x10 10 particles) or PBS as a control. After 6 days of co-culture, cells were harvested and stained with anti-CD3-PE/Cy7, anti-CD4-APC, anti-CD8-APC-H7 antibodies and 7AAD (BD Biosciences). Proliferation and cell death of T-cell subsets were monitored by FACS.
In assays determining the immunomodulatory effects of CPC and EV-CPC, HLA-mismatched CFSE-labeled PBMC (1x10 5 cells) were stimulated with 1 μg/mL phytohemagglutinin (Sigma-Aldrich) in the presence or absence of mitomycin-C-treated CPC (at CPC: PBMC ratios of 1:5 and 1:10) or with increasing concentrations of EV (EV1: 5.5x10 8 , EV2:5.5x10 9 or EV3: 1.7x10 10 particles) for 5 days. At the end of the co-culture, the proliferation and death rate of the T cells were monitored by FACS.
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