The ploidy of the triploid hybrids obtained from the two combinations was validated through both chromosome preparation and flow cytometry analyses. The method for chromosome preparation was that proposed by Wen et al. (2020) [75 (link)]. Briefly, root-tip tissues with a length of about 1 cm were treated with 0.002 mol/L 8-hydroxyquinoline aqueous solution for 4 h and then fixed in Carnoy’s solution overnight. Subsequently, the apical meristems were cut into the size of about 1 mm3 for the following enzymolysis. Next, the enzyme-treated apical meristems were removed from Carnoy’s solution for final chromosome preparation on a slide. The chromosomes were stained with 5% Giemsa and visualized under a microscope (Olympus, Tokyo, Japan). Flow cytometry analyses were conducted as was described by Galbraith et al. (1983) [76 (link)]. The stain method used in this study was proposed by Miller et al. (2012) [77 (link)]. A BD Accuri C5 flow cytometer (BD Bioscience, San Jose, CA, USA) was adopted for the flow cytometry analyses in this study.
Accuri c5 flow cytometer
Manufactured by BD
Sourced in United States
The BD Accuri C6 flow cytometer is a compact and user-friendly instrument designed for analyzing and sorting cells. It utilizes laser-based technology to measure and characterize the physical and fluorescent properties of individual cells flowing through the instrument. The core function of the BD Accuri C6 is to provide accurate and reliable data on cell population size, composition, and other key characteristics.
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Lab products found in correlation
Accuri c6, by BD (5 mentions)
Cellquest pro software, by BD (3 mentions)
Apo direct kit, by BD (2 mentions)
Daf 2da, by Merck Group (1 mentions)
Trypsinization, by Thermo Fisher Scientific (1 mentions)
Anti cd3 antibody okt3, by BioLegend (1 mentions)
Accuri, by BD (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
Affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Fixation buffer, by R&D Systems (1 mentions)
Permeabilization buffer, by R&D Systems (1 mentions)
Fitc conjugated anti human monoclonal antibody t bet, by R&D Systems (1 mentions)
Pe conjugated anti human monoclonal gata3, by R&D Systems (1 mentions)
Annexin 5 fluorescein isothiocyanate apoptosis detection kit, by BD (1 mentions)
Annexin 5 fitc, by BioLegend (1 mentions)
Caspglow fluorescein active caspase staining kit, by Abcam (1 mentions)
Anti cd45 pe, by BioLegend (1 mentions)
Anti cd86 pecy5, by BioLegend (1 mentions)
Anti f4 80 pe, by BioLegend (1 mentions)
22 protocols using accuri c5 flow cytometer
1
Ploidy Validation of Triploid Hybrids
2
Quantification of Intracellular NO and ROS
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To quantify NO, cell suspensions were treated with DAF2DA (2 μM) (Sigma-Aldrich, D225, São Paulo, Brazil) and incubated for 1 h at 37°C and 5% CO2. After labeling, samples were stored at 4°C in the dark until analysis using a flow cytometer (BD Accuri™ C5 flow cytometer), and the data were analyzed using BD Accuri™ C6 software (version 1.0.264.21). Cells (1 × 105/mL) from various experimental groups were evaluated by fluorescence at 10,000 events. Cells without labeling were used as a negative control to delimit the negative populations. After excluding the debris, the cellular fluorescence of the triazole product (DAF-2T) was collected in the FL1 channel, and myeloid cells were gated separately. The mean values of FL1 were used for NO quantification.
To measure ROS levels, 10 µM H2DCFDA (Introgen-Leiden Molecular Probes) was added to cell suspensions and incubated for 1 h at 37°C and 5% CO2. Phorbol 12-myristate 13-acetate (PMA, 10 µM) was added 30 min before analysis for the positive ROS control. After labeling, the ROS analysis procedure was identical for NO.
To measure ROS levels, 10 µM H2DCFDA (Introgen-Leiden Molecular Probes) was added to cell suspensions and incubated for 1 h at 37°C and 5% CO2. Phorbol 12-myristate 13-acetate (PMA, 10 µM) was added 30 min before analysis for the positive ROS control. After labeling, the ROS analysis procedure was identical for NO.
3
Apoptosis Induction by Compound G5
4
Detecting Autophagic Vesicles with Acridine Orange
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Autophagy is characterized by the formation of acidic vesicular organelles (AVOs). To detect AVOs, cells can be stained with acridine orange, a nucleic acid-specific fluorescent cationic dye (21 (link)). The cells were seeded at a density of 2×105 cells in six-well plates and allowed to attach. Subsequent to treatment with 0.8 mg/ml SJKJT for 6 h at 37°C, the cells were stained with 1 µg/ml acridine orange for 10 min at 37°C, collected by trypsinization (Gibco Life Technologies) and resuspended in PBS. The green (510–530 nm) and red (650 nm) fluorescence, which was emitted from 1×104 cells illuminated with blue (488 nm) excitation light, were measured using a BD accuri C5 flow cytometer and BD Accuri™ C6 version 1.0.264.21 software (BD Biosciences, Franklin Lakes, NJ, USA).
5
Binding of Anti-LAG3 Antibodies to Human T Cells
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Example 13
Anti-LAG3 antibodies (Clone 2#, 8#, 13#) were tested for the ability of binding to human LAG-3 expressed on activated human T cells.
Primary T cells were isolated from peripheral blood mononuclear cells with magnetic beads and cultured in tissue culture plates coated with anti-CD3 antibody (OKT3, Biolegend). Anti-LAG-3 antibodies (Clone 2#, 8#, 13#) and negative control IgG4 were added to cells and the mixture was incubated at 4° C. for 30 minutes. The cells were washed twice. The binding activity of the anti-LAG-3 antibodies to LAG-3 expressed on T cells was detected using an R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch) secondary reagent, with the mixture incubated at 4° C. for 30 minutes followed by washing twice. Then, cells were resuspended in PBS buffer. Analysis of LAG-3 binding was carried out with the BD Accuri C5 flow cytometer (BD Bioscience).
Representative curves for these clones binding to LAG-3 expressed by human T cells were shown in
6
Apoptosis Evaluation of Naringenin-Treated Cells
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The Annexin V/propidium iodide (PI) double staining assay was performed to examine cell apoptosis by using the Annexin V/PI detection kit (cat. no. PF00005; Proteintech Group, Inc., Rosemont, IL, USA). HOS and U2OS cells were treated with naringenin at various concentrations (100, 250, and 500 μM) for 24 h. After treatment, cells were collected and washed with PBS twice and then resuspended in a staining buffer containing PI and Annexin V–FITC at room temperature for 30 min; cells were placed in the dark prior to flow cytometry. Cells were analyzed using the BD Accuri C5 flow cytometer and BD Accuri C6 software (version 1.0.264.21, BD Biosciences).
7
Flow Cytometry Calibration and Analysis
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Flow cytometry experiments were performed in a BD Accuri C5® Flow
Cytometer (USA) equipped with a 488-nm laser for fluorescence excitation. For each
sample, 100,000 events were acquired, and the median fluorescence intensities were
obtained from histograms of FL2-H 585/40 nm channel. Flow cytometry calibration was
performed using spherothech 8-peak beads (BD Accuri™, USA) according to
the manufacturer's recommendations and instructions.
Cytometer (USA) equipped with a 488-nm laser for fluorescence excitation. For each
sample, 100,000 events were acquired, and the median fluorescence intensities were
obtained from histograms of FL2-H 585/40 nm channel. Flow cytometry calibration was
performed using spherothech 8-peak beads (BD Accuri™, USA) according to
the manufacturer's recommendations and instructions.
8
Internalization of Anti-LAG-3 Antibodies
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Example 7
Anti-LAG-3 antibodies were tested for the ability to be internalized on Jurkat-LAG-3 cells.
Jurkat-LAG3 cells transfected with human LAG3 gene and thus stably expressing human LAG-3 were incubated with anti-LAG-3 antibodies (LAG3 2# and LAG3.5(BMS)) in duplicates for 1 hour at 4° C. The cells were washed once, divided into 2 groups, one of which incubated at 37° C. and the other incubated at 4° C. After 2 hours, the binding was detected using a FITC conjugated AffinityPure Donkey Anti-human (H+L) IgG (Jackson Immuno Research) secondary reagent incubated at 4° C. for 30 min followed by washing once. After that, cells were resuspended in PBS buffer. Analysis of human LAG-3 binding was carried out with the BD Accuri C5 flow cytometer (BD Bioscience).
As shown in
9
Quantifying Apoptosis in Cancer Cells
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Apoptotic cells were quantified using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which examines breaks in DNA strands caused during cell apoptosis by using the BD APO-DIRECT kit (BD Biosciences, San Jose, CA, USA; cat. no. 556381). HOS and U2OS cells (2 × 106) were treated with naringenin at various concentrations (100, 250, and 500 μM) for 24 h. After treatment, cells were collected and centrifuged at 950× g for 10 min at 4°C. Cells were fixed with 1% paraformaldehyde for 30 min on ice and washed with PBS twice. After the removal of the fixative, 0.5 mL of ethanol was added, and the mixture was incubated at −20 °C for 4 h. Subsequently, ethanol was removed through centrifugation and cellular DNA was obtained. The cellular DNA was stained with TUNEL solution (3 ng/mL TdT enzyme and 0.04 nmol FITC dUTP) at 37 °C for 1 h. After incubation with TUNEL solution, cells were washed with a rinse buffer (1 mL; BD APO-DIRECT kit; BD Biosciences; cat. no. 556381) and centrifuged at 1425× g for 10 min at 4 °C. The fluorescein-labeled DNA strand was detected and quantified using the BD Accuri C5 flow cytometer and BD Accuri C6 software (version 1.0.264.21, BD Biosciences).
10
Blocking Phosphatidylserine-TIM-3 Interaction
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Example 10
Phosphatidylserine-TIM-3 interaction blocking assay was performed as follows.
Briefly, Jurkat T cells (CBTCCCAS, Clone E6-1) were incubated with 1 μg/mL anti-human CD95 (Fas) antibody (Clone E059.1, Biogems, Cat #08011-25-500) for 16 h. When Jurkat T cell were induced to undergo apoptosis, phosphatidylserines flipped to the extracellular surface of the cell, to which TIM-3 might bind.
Human TIM-3-mFc protein (amino acid sequence set forth in SEQ ID NO: 55) of 25 μl (40 μm/ml) was mixed and incubated with 25111 of serially diluted antibodies (started at 1 μm/mL) in Annexin V binding buffer (Biolegend Cat 422201) at room temperature (RT) for 20 minutes. Then, the mixture was added to 2×105 Jurkat T cells in 50 μl binding buffer (PBS containing 0.5% BSA). After incubation at 4° C. for 40 minutes, the cells were pelleted (3 minutes, 600×g), washed once using binding buffer with 0.5% BSA and re-pelleted. The cells were then added with PE conjugated AffiniPure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), Fcγ Fragment Specific (Jackson ImmunoResearch, Cat #115-115-164) diluted at 1:100, and were analyzed with the BD Accuri C5 flow cytometer.
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