For colony formation assay, indicated cells were seeded in a 6-well plate (China, NEST, Cat. 703001) with 600 cells per well supplemented with 2 mL cell culture medium, and the cell culture medium was changed every 3 days for 2~3 weeks, and then indicated cells were fixed with 4% PFA and stained with 0.5% crystal violet. Annexin V FITC Apoptosis Detection Kit I (556547, BD, China) was used to evaluate the cellular apoptosis according to the manufacturer’s instructions. For cell cycle analysis experiments, indicated cells were digested and washed with PBS twice and then fixed in 75% alcohol overnight at -20°C. The fixed cells were washed three times and then stained with propidium iodide (PI) staining buffer at room temperature for 30 min in the dark, and then the cell cycle was analyzed by the FACSAria SORP machine (BD, USA).
Facsaria sorp machine
Manufactured by BD
Sourced in United States
The FACSAria SORP machine is a flow cytometry instrument designed for cell sorting. It is capable of analyzing and sorting various types of cells and particles based on their physical and fluorescent properties.
Automatically generated - may contain errors
Lab products found in correlation
Fitc annexin 5 apoptosis detection kit 1, by BD (4 mentions)
Cd19 bv605, by BD (2 mentions)
Cd27 pecf594, by BD (2 mentions)
Igd bv421, by BD (1 mentions)
Cd21 pe cy7, by BioLegend (1 mentions)
Cd38 percp cy5, by BioLegend (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Compensation beads, by BD (1 mentions)
Igm bv570, by BioLegend (1 mentions)
Cd14 efluor 605nc, by Thermo Fisher Scientific (1 mentions)
Cd43 apc, by Thermo Fisher Scientific (1 mentions)
Cd14 fitc, by Immunotools (1 mentions)
Neutravidindylight 650, by Thermo Fisher Scientific (1 mentions)
Recombinant rnasin ribonuclease inhibitor, by Promega (1 mentions)
96 well pcr plate, by Eppendorf (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Cd3 fitc, by Immunotools (1 mentions)
24 well plate chamber insert, by Corning (1 mentions)
Pi rnase staining buffer, by Cell Signaling Technology (1 mentions)
Facsdiva 8, by BD (1 mentions)
7 protocols using facsaria sorp machine
1
Colony Formation, Apoptosis, and Cell Cycle Analysis
2
Multi-color Flow Cytometry Immune Profiling
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PBMCs were stained immediately after thawing in FACS buffer (PBS with 0.1% NaN3 and 2% FBS; Lonza). Color compensations were done using a mix of cells and compensation beads (BD Biosciences). After incubation of the cells with antibodies on ice for 30 min, they were washed with FACS buffer. Hoechst (Invitrogen) live/dead marker was added shortly prior to measuring on a FACSAria SORP machine (BD Biosciences). The following antibodies were used in the panel: CD14-eFluor605NC, CD24-eF450, CD43-APC, CD23-APC-eFluor780 (eBioscience), IgD-BV421, CD19-BV605, IgG-PE (BD Pharmingen), CD10-BV510, CD138-BV711, CD27-PECF594 (BD Horizon), IgM-BV570, CD38-PerCP-Cy5.5, CD21-PE-Cy7, CD20-AF700 (BioLegend), CD5-FITC, CD3-PE-Dy647 (Immunotools).
3
Annexin V FITC Apoptosis Assay
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The Annexin V FITC Apoptosis Detection Kit I (556547; BD, Shanghai, China) was used to evaluate cellular apoptosis according to the manufacturer’s instructions. For cell flow cytometry experiments, the indicated cells were digested and washed with PBS twice and then fixed in 75% alcohol overnight at −20°C. The fixed cells were washed three times and then stained with propidium iodide (PI) buffer at room temperature for 30 min in the dark. The cell cycle was then analyzed using the FACSAria SORP machine (BD, San Diego, CA, United States).
4
Sorting Antigen-Specific Memory B Cells
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To sort antigen-specific cells, tetramers were generated as described in Franz et al. (52 (link)). A mix of Neutravidin (Neutravidin DyLight650, Thermo Scientific) and biotinylated peptide was incubated in the dark on ice for 30 min. Aggregates were removed by high speed centrifugation. B cells from acute Borreliosis patients were stained for 30 min with a pool of the three peptide tetramers and washed twice with FACS buffer (PBS, 2% FBS). The following antibodies were used to distinguish the different memory B cell subpopulations and to gate out monocytes, T-cells and dead cells: CD14-FITC, CD3-FITC (Immunotools), IgD-BV421, CD27-PECF594 (BD Horizon), CD19-BV605 (BDPharmingen), and live/dead marker. CD20-Biotin (Immunotools) was used as a compensation control for Neutravidin. Single cells were sorted on a FACSAria SORP machine (BD Biosciences) into 96-well PCR plates (Eppendorf) containing 5μl of 0.5% PBS, 10 mM DTT (Invitrogen), and 5U Recombinant RNasin® Ribonuclease Inhibitor (Promega) per well (53 (link)). The plate holder of the sorter was kept at 4°C throughout the sorting procedure. Random, negative and tetramer positive CD19+CD27+CD14−, CD3−, Hoechst− B cells were sorted into 96-well plates, which were immediately put on dry ice and stored at −80°C.
5
Apoptosis and Cell Cycle Analysis
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Annexin V FITC Apoptosis Detection Kit I (556547, BD, Guangzhou, China) was used to evaluate the cellular apoptosis according to the manufacturer’s instructions. For cell cycle analysis experiments, the indicated cells were digested and washed with PBS twice and then fixed in 75% alcohol overnight at −20°C. The fixed cells were washed three times and then stained with propidium iodide (PI) staining buffer at room temperature for 30 min in the dark, and then the cell cycle was analyzed using the FACSAria SORP machine (BD, USA).
6
Comprehensive Cell Migration and Apoptosis Assays
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Cell migration assays was performed as previously documented. Briefly, to produce a wound, the monolayer cells in 6-well plate were scraped in a straight line with pipette tips. Plate was then washed with PBS to remove detached cells. Photographs of the scratch were taken at indicated time points using Nikon inverted microscope (Ti-S). Gap width was calculated using GraphPad Prism software. For trans-well assay, 2.5×104 cells in 100μL serum free medium were plated in 24-well plate chamber insert (Corning Life Sciences, Cat. 3422), with the medium containing 10% FBS at the bottom of the insert. Cells were incubated for 24 h, and then fixed with 4% paraformaldehyde for 20 min. After washing, cells were stained with 0.5% crystal violet blue. The positively stained cells were pictured and counted. Annexin V FITC Apoptosis Detection Kit I (556547, BD, China) was used to evaluate the cellular apoptosis according to the manufacturer’s instructions. For cell cycle analysis experiments, indicated cells were digested and washed with PBS twice and then fixed in 75% alcohol overnight at − 20 °C. The fixed cells were washed three times and then stained with propidium iodide (PI) staining buffer at room temperature for 30 min in the dark, and then the cell cycle was analysed by FACSAria SORP machine (BD, USA).
7
Multiparametric Flow Cytometry Analysis of Cellular Organelles
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HeLa, HepG2, and HCT116 cells were costained with LysoTracker Red (0.3 µM) and Hoechst 33342 for 30 min. After extensive washing, cells were suspended in cold PBS and analyzed by flow cytometry. For LAMP1 immunostaining, cells were first fixed with 4% PFA for 15 min and permeabilized with permeabilization buffer (1× PBS, 0.3% Triton X-100, and 0.5% BSA) for 15 min and incubated with LAMP1 antibody in the incubation buffer (0.5% BSA in PBS) for 1.5 h at room temperature. After extensive washing, cells were resuspended in incubation buffer containing Alexa Fluor 488 secondary antibody for 30 min. Cells were then collected and incubated in PI/RNase staining buffer (4087; Cell Signaling Technology) for an additional 30 min and analyzed by flow cytometry. For the FITC-Dextran experiment, cells were fed with 0.25 mg/ml FITC-Dextran for 12 h followed by 2-h recovery in fresh DMEM medium. Cells were then fixed with 70% ethanol (2 h, 4°C) and stained with PI for 30 min and analyzed by flow cytometry. All samples were analyzed on a FACSAria SORP machine (BD Biosciences), and the fluorescence intensity profiles of each cell cycle stage were analyzed using BD FACSDiva 8.0.1. Cell cycle distribution was analyzed by PI or Hoechst 33342 content and fitted to the Dean-Jett-Fox cell cycle model using FlowJo software.
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