The compounds (Online Supplementary Table S2) were dispensed on 96-well V-bottom plates (Thermo Fisher Scientific, Carlsbad, CA) and 384-well plates (Corning, Corning, NY, USA) using an acoustic liquid handling device Echo 550 (Labcyte, Sunnyvale, CA). Drug plate layouts and concentrations are presented in Online Supplementary Figure S1. BM mononuclear cells (BM-MNC) were isolated using Ficoll-Paque Premium (GE Healthcare, Little Chalfont, Buckinhamshire, UK) density gradient centrifugation. Fresh or frozen BM-MNC were suspended in mononuclear cell medium (MCM; PromoCell, Heidelberg, Germany) supplemented with 10 μg/mL gentamicin and 2.5 μg/mL amphotericin B and plated in parallel on pre-drugged 96-well plates (100,000 cells/well in 100 μl) for FC analysis and 384-well plates (10,000 cells/well in 25 μl) for CellTiter-Glo® (CTG)-based cell viability assay. The cells were incubated with the drugs for 72 hours at 37°C and 5% CO2.
96 well 5 bottom plates
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 96-well V-bottom plates are a type of microplate designed for various laboratory applications. These plates feature a V-shaped well bottom, which can facilitate efficient sample mixing and settling. The 96-well format provides a standardized layout for high-throughput sample processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fixation permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Rhil 3, by Miltenyi Biotec (2 mentions)
Aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Phospho flow pe mouse anti p stat5 py694 antibody, by BD (2 mentions)
Ficoll hypaque density centrifugation, by Cytiva (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Echo 550, by Beckman Coulter (1 mentions)
384 well plate, by Corning (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
Lsr4 laser, by BD (1 mentions)
Fluorofix buffer, by BioLegend (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
Brilliant stain buffer plus, by BD (1 mentions)
Rhgm csf, by Miltenyi Biotec (1 mentions)
Cd4 fitc, by Sony (1 mentions)
5 protocols using 96 well 5 bottom plates
1
High-throughput screening of myeloid leukemia cells
2
PBMC Isolation and Phospho-STAT5 Analysis
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Human peripheral blood mononuclear cells (PBMCs) from a healthy donor were isolated from whole blood by Ficoll- Hypaque density centrifugation (Amersham-Pharmacia- Biotech). The cells were counted and plated at 2 × 106 cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 μL of RPMI (GibcoBRL, Invitrogen) supplemented with 10% fetal bovine serum (GibcoBRL, Invitrogen), or 100 μL of RPMI supplemented with 1:10 serafrom patients or controls. PBMCs were left unstimulated or were stimulated with 10 ng/μL of rhIL-3 or GM-CSF or 50 ng/μL of rhIL-3 (Miltenyi-Biotec) for 15 min at 37°C. Thereafter, cells were fixed and permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with antibodies anti CD14-Pacific Blue and anti CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific). STAT5 phosphorylation (p-STAT5) levels were assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).
3
SARS-CoV-2 Spike Protein Binding Assay
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The SFB assay was performed according to previously described methods (16 (link), 17 (link)). In brief, cells expressing the spike protein of the ancestral Wuhan strain, Omicron BA.1 and XBB were seeded at 1.5 x 105 cells/well in 96-well V-bottom plates (Thermo Fisher Scientific, Waltham, USA). The cells were incubated with human serum (diluted 1:100 in 10% FBS; HyClone, Chicago, USA), followed by a second incubation with a double stain comprising Alexa Fluor 647-conjugated anti-human IgG (diluted 1:500; Thermo Fisher Scientific) and propidium iodide (PI; diluted 1:2500; Sigma-Aldrich, Burlington, USA). Cells were acquired using an LSR4 laser (BD Biosciences, New Jersey, USA) and analyzed using FlowJo (Tree Star, BD Biosciences). The percentage of GFP-positive spike protein-expressing cells bound by the antibody, indicated by Alexa Fluor 647- and FITC-positive events, was used as an indicator of binding. The assay was performed as two independent experiments, each with technical duplicates. The amount of spike protein expressed on the cell surface was verified by ACE-2-HuFc binding. A subset of age-matched samples was randomly selected and examined for binding antibodies against Omicron BA.1 and XBB (n = 10 per LTRs with double and triple IS regimens and HC).
4
PBMC Immunophenotyping by Flow Cytometry
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Approximately 1.1 × 107 PBMCs were washed with D-PBS, suspended in 275 µL of
Zombie Aqua (BioLegend, San Diego, CA, USA) diluted 100-fold in D-PBS, and kept on ice
for 15 min. In addition, 275 µL of TruStain FcX (BioLegend) diluted 10-fold in D-PBS was
added, and the mixture was gently vortexed and kept on ice for 15 min. After the
reaction, 50 µL (approximately 1.0 × 106 cells) of the cell suspension was
dispensed into 96-well V-bottom plates (Thermo Fisher Scientific, Waltham, MA, USA) for
mean fluorescence intensity (MFI) analysis, and the same volume of antibody diluent was
added to each plate and mixed with the cell suspension. The combinations of antibodies
and dilution factors are shown in Supplementary Table 1. Brilliant Stain Buffer Plus (BD
Biosciences, San Jose, CA, USA) was added to the antibody mixture. The plates were then
incubated on ice for 30 min. After washing with 2% FBS/PBS, the cells were fixed with
200 µL FluoroFix Buffer (BioLegend) and allowed to react for 30 min under light
shielding at RT. After centrifugation (460 × g, 5 min, 20°C), 200 µL of 2% FBS/PBS was
added to each well, and the cells were stored at 4°C until FCM analysis.
Zombie Aqua (BioLegend, San Diego, CA, USA) diluted 100-fold in D-PBS, and kept on ice
for 15 min. In addition, 275 µL of TruStain FcX (BioLegend) diluted 10-fold in D-PBS was
added, and the mixture was gently vortexed and kept on ice for 15 min. After the
reaction, 50 µL (approximately 1.0 × 106 cells) of the cell suspension was
dispensed into 96-well V-bottom plates (Thermo Fisher Scientific, Waltham, MA, USA) for
mean fluorescence intensity (MFI) analysis, and the same volume of antibody diluent was
added to each plate and mixed with the cell suspension. The combinations of antibodies
and dilution factors are shown in Supplementary Table 1. Brilliant Stain Buffer Plus (BD
Biosciences, San Jose, CA, USA) was added to the antibody mixture. The plates were then
incubated on ice for 30 min. After washing with 2% FBS/PBS, the cells were fixed with
200 µL FluoroFix Buffer (BioLegend) and allowed to react for 30 min under light
shielding at RT. After centrifugation (460 × g, 5 min, 20°C), 200 µL of 2% FBS/PBS was
added to each well, and the cells were stored at 4°C until FCM analysis.
5
Quantifying STAT5 Phosphorylation in PBMC Subsets
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Human peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated from whole blood by Ficoll-Hypaque density centrifugation (Amersham-Pharmacia-Biotech, Sweden). The cells were counted and plated at 2 × 106 cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 µL RPMI (GibcoBRL, Invitrogen), supplemented with 10% fetal bovine serum (FBS) (GibcoBRL, Invitrogen) or 100 µL RPMI supplemented with 10% plasma from patients or controls. PBMCs were left unstimulated or were stimulated with 5 to 80 ng/mL rhGM-CSF or 100 ng/mL rhIL-3 (Miltenyi-Biotec) for 30 min at 37°C, and the cells were then fixed permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with CD14-Pacific Blue and CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific), and STAT5 phosphorylation (p-STAT5 levels) was assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD-Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).
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