With exposure to H2O2 for 24 h, apoptotic and necrotic appearances were evaluated using a flow cytometry with annexin V-FITC and propidium iodide (PI) staining (Annexin V-FITC Kit: Beckman Coulter, CA, USA). This staining is based on the principles that annexin V binds to phosphatidylserine expressed on the surface of apoptotic cells and that membrane-impermeable PI is incorporated into the DNA of necrotic cells. Both floating and attached cells were collected into a tube. The cell suspension were treated with 25 μL of annexin V-FITC and 12.5 μL of PI (6.25 μg/mL) for 10 min in the dark on ice. After being filtered with a cell strainer, the cell suspension was analyzed using a FACS Aria II system (Becton Dickenson, Franklin Lakes, NJ, USA).
Facsaria 2 system
Manufactured by BD
Sourced in United States
The FACSAria II system is a high-performance cell sorter designed for advanced flow cytometry applications. It offers precise cell sorting capabilities and advanced fluidics technology to ensure accurate and reliable results.
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Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Facsdiva software, by BD (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Reserpine, by Merck Group (3 mentions)
Ctla 4, by BD (2 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Cd3 fitc clone17a2, by BioLegend (2 mentions)
Cd8 percp cy5 5 clone 53 6, by Thermo Fisher Scientific (2 mentions)
Cd45 bv510 clone 30 f11, by BioLegend (2 mentions)
Cd11b pe dazzle594 clone m1 70, by BioLegend (2 mentions)
Cd68 fluorescein isothiocyanate, by Thermo Fisher Scientific (2 mentions)
Ki67 allophycocyanin, by BioLegend (2 mentions)
Il 32 alexa fluor 488, by R&D Systems (2 mentions)
Cd14 efluor450, by Thermo Fisher Scientific (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Blasticidin, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
110 protocols using facsaria 2 system
1
Apoptosis and Necrosis Evaluation
2
Cardiomyocyte Differentiation of ESCs
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Mouse ESCs were cultured in accordance with previous studies and adapted to gelatin-coated dishes in the presence of leukemia inhibitory factor (LIF) for 2 days prior to differentiation32 (link). CM differentiation of ESCs was performed per a previously-published protocol21 (link). On the day of flow sorting, embryonic bodies (EBs) were digested with trypsin/EDTA for 3 minutes followed by collagenase A + B solution (10 mg/ml each) for 30 minutes. These cells were then re-suspended in HBSS (Life Technologies) with 20% FCS (Sigma). The eGFP-expressing cells were analyzed and sorted using a FACS Aria II system, and flow cytometry data were analyzed using FACS Diva Software (Becton Dickinson).
3
Comprehensive Immune Cell Profiling by Flow Cytometry
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To examine differences in cell profiles before and after the treatment with DAAs, a flow cytometry analysis was performed. Isolated PBMCs, which were the same as the samples used for the ELIPOST assay, were stained by different antibodies and then examined using flow cytometry with the Becton Dickinson FACSAria II system. Based on previously reported data, a FACS analysis was performed using the following antibodies: anti-CD45RA, Foxp3, CD3, CD4, CD8, PD-1, CTLA-4, CD25, CCR6, CXCR3, CCR4, 4-1BB, OX40, and CD80 (BD Biosciences, Franklin Lakes, NJ, USA) [7 (link),8 (link),17 (link),22 (link)]. The antibody CD45RA was used to divide CD4 or CD8 into naïve and memory T cells. Intracellular staining with anti-Foxp3 was conducted to characterize regulatory T cells (Tregs). In addition, the cell profiles of MDSCs were analyzed using anti-CD14, CD15, CD33, CD11b, HLA-DR, and PD-L1.
4
Purification and Identification of Mouse Brain Nuclei
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Mouse brain samples were processed as described74 . Briefly, brains were suspended in lysis buffer (0.32 M sucrose; 0.1 mM ethylenediaminetetraacetic acid; 5 mM CaCl2; 3 mM Mg(Acetate)2; 10 mM Tris–HCl pH 8; 1 mM dithiothreitol; and 0.1% Triton X-100) and processed using a 15 ml glass dounce homogenizer (Wheaton, UK) in 5–10 strokes on ice. Samples were centrifuged at 100,000xg with a two-step sucrose gradient to purify nuclei. Nuclei were stained with primary antibody NeuN (Abcam, EPR12763 1:250) and secondary antibody Alexa-488 (Abcam, ab150077, 1:1000). All nuclei were also stained with Hoechst 33342 (SIGMA, USA). Samples were sorted using the Becton Dickinson FACSAria II system.
5
Isolation and Characterization of Human Platelets
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Platelets were isolated by differential centrifugation as previously reported
19 (link) with some modifications. Briefly, human blood was collected from healthy volunteers who had not received any medications in the last 2 weeks. The blood was transferred to tubes containing 1/9 volume of anticoagulation citrate‐dextrose solution (Sigma‐Aldrich), and centrifugation at 100 g was carried out for 20 min to obtain PRP. The PRP was centrifuged at 100 g for 15 min to pellet red and white blood cells, and the supernatants were centrifuged at 800 g for another 15 min to obtain platelets and quantified using a hemacytometer. A portion of platelets was incubated with APC antihuman CD61 Ab (B312450; BioLegend), followed by purity analysis using the FACSAria II system (Becton Dickinson). The APC mouse IgG1 κ isotype control (B344277; BioLegend) was used as the isotype control (FigureS1–S2 ). Another portion of the platelets was stained using PKH67 Green Fluorescent Cell Linker Kits (Sigma‐Aldrich) according to the manufacturer's protocol and used in the biological assays.
19 (link) with some modifications. Briefly, human blood was collected from healthy volunteers who had not received any medications in the last 2 weeks. The blood was transferred to tubes containing 1/9 volume of anticoagulation citrate‐dextrose solution (Sigma‐Aldrich), and centrifugation at 100 g was carried out for 20 min to obtain PRP. The PRP was centrifuged at 100 g for 15 min to pellet red and white blood cells, and the supernatants were centrifuged at 800 g for another 15 min to obtain platelets and quantified using a hemacytometer. A portion of platelets was incubated with APC antihuman CD61 Ab (B312450; BioLegend), followed by purity analysis using the FACSAria II system (Becton Dickinson). The APC mouse IgG1 κ isotype control (B344277; BioLegend) was used as the isotype control (Figure
6
Cell Cycle Profiling by Flow Cytometry
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Cell cycle analysis was performed as previously described22 (link). In brief, trypsinized cells at a density of 106 cells/ml in phosphate buffered saline (PBS) were fixed by 70% ethanol. Ethanol-fixed cells were stained in the staining buffer made of propidium iodide (0.4 mg/ml), RNase A (0.1 mg/ml) and 0.1% Triton X-100 in PBS. The cell-cycle profile and sub-G1 fraction were analyzed by flow cytometry using the Becton Dickinson FACSAria II system (Franklin Lakes, NJ).
7
Multicolor FACS for Immune Profiling
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To determine the frequency of immune cells, multi‐color fluorescence‐activated cell sorting (FACS) analysis was performed using the following antibodies: anti‐CD3, CD4, CD8, CD14, CD15, CD25, CD80, CD45RA, HLA‐DR, FoxP3, CTLA‐4, PD‐1, PD‐L1, CCR4, CCD6, CXCR3, 4‐1BB, and OX40 (Becton Dickinson). Flow cytometry was done using the Becton Dickinson FACSAria II system.
8
Immune Profiling of Peripheral Blood Cells
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For the analysis of ICP in peripheral blood, PBMCs of 41CCA, 10 HV and 20 HCV SVR12 patients were used. CCA with cholangitis underwent biliary drainage to improve cholangitis. PBMCs before treatments, such as surgery and chemotherapy, were used for 41 patients. For patients who underwent surgery, ICP of PBMCs after surgery were also investigated. PBMCs after discharge with improvement of postoperative complications were used as postoperative PBMCs. For patients who underwent chemotherapy, ICP of PBMCs after chemotherapy were not investigated. For HCV SVR12 patients, PBMCs at SVR12 were used. To measure the frequency of immune cells, FACS analysis using the FACSAria II system (Becton Dickinson, Franklin Lakes, NJ, USA) was performed employing the following antibodies: CD3, CD4, CD8, CD14, CD15, CD25, CD80, CD45RA, human histocompatibility leukocyte antigen (HLA)-DR, forkhead box P3 (FoxP3), cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death-1 (PD-1), PD-1 ligand (PD-L1), C-C chemokine receptor (CCR)4, CCR6, C-X-C motif chemokine receptor 3 (CXCR3), 4-1BB, and OX40 (Becton Dickinson).
9
Tetramer-Based Quantification of Antigen-Specific CTLs
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Peptide MRP3 765 -specific tetramer was used for the detection of peptide Mizukoshi et al. Phase I Trial of MRP3-Derived peptide for HCC. vaccine-induced CTLs. Peptide HIVenv 584 -specific tetramer was used as a negative control of tetramer assay. All tetramers were purchased from Medical Biological Laboratories Co., Ltd. (Nagoya, Japan). PBMCs were stained with anti-CD8-APC (Becton Dickinson, Tokyo, Japan), anti-CCR7-FITC (eBioscience, Tokyo, Japan), anti-CD45RA-PerCP-Cy5.5 antibodies (eBioscience, Tokyo, Japan), and with tetramer-PE for 30 min at room temperature. Cells were washed, fixed with 0.5% paraformaldehyde/PBS, and analyzed using a Becton Dickinson FACSAria II system. In the tetramer assays with the negative control tetramer, we did not observe more than 0.03% tetramer-positive cells in any assay. Based on the results of the negative control, responses to MRP 765 -specific tetramer were considered positive if more than 0.03% tetramer positive cells were detected. At least 1,000,000 PBMCs were acquired for each tetramer assay. For the detection of Tregs and MDSCs, the following anti-human monoclonal antibodies were used: anti-CD4 (Becton Dickinson), anti-CD14 (Becton Dickinson), anti-CD25 (Becton Dickinson), anti-CD127 (Becton Dickinson), and anti-HLA-DR (Becton Dickinson).
10
Tumor Cell Isolation and Analysis
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Tumors were enzymatically digested with collagenase type I (1.6 kU/mL; Sigma) St. Louis, MO, USA and DNase type I (1.5 kU/mL; Sigma). Digested tumor suspension was stained with PE-conjugated anti-CD11b antibody (BD Bioscience), PE-conjugated anti-F4/80 antibody (AbD Serotech), or PE-conjugated anti-CD31 antibody (BioLegend), and analyzed by using a FACS Aria II system (BD Bioscience). Cells were gated according to forward/side scatter, and dead cells were excluded by using propidium iodide (Sigma) staining. In some experiments, cells were sorted from the mixture of all the tumors in each group. The whole RNA was extracted, and cDNA was obtained by using a Cells-to-CT kit (Applied Biosystems) Foster City, CA, USA for quantitative PCR analysis.
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