The kit CellROX® Deep Red (Invitrogen, Waltham, MA, USA) for flow cytometry assay was used for the determination of ROS and the dye (4-amino-5-methylamino-2′,7′-difluorofluorescein) diacetate (DAF-FM DA) (Thermo Scientific, Eugene, OR, USA) was used to determine RNS. The fluorescent probes were used at a 5 µM concentration for labeling purposes. For this reason, the macrophages, bacteria and supernatants were suspended in a 98 µL final volume [26 (link),27 (link)]. The labeling procedure was started 60 min before the end of the incubation time for each assay. First, the CellROX® Deep Red (5 µM) probe was added and the tubes were incubated for 30 min at 37 °C in 5% CO2 in the dark. After that, the DAF-FM DA probe (5 µM) was added to the tubes and they were incubated for another 30 min. A separated tube of macrophages with the same assay conditions were labeled with 4 μL of conjugated-APC monoclonal antibody anti-mouse F4/80 (Thermo Scientific, Eugene, OR, USA), incubating this for 30 min in darkness [28 (link)]. The analysis was performed on a BD FACSLyric™ Flow Cytometry System (Figure 1 ).
Facslyric flow cytometry system
Manufactured by BD
Sourced in United States
The BD FACSLyric Flow Cytometry System is a laboratory instrument designed for the analysis and sorting of cells. It utilizes flow cytometry technology to identify and quantify various cell types and their characteristics within a sample. The system is capable of detecting and measuring multiple parameters of individual cells as they pass through a laser beam.
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Lab products found in correlation
Daf fm da, by Thermo Fisher Scientific (1 mentions)
Cellrox deep red, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg conjugated with alexa 488, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Cd86 pe cy7, by Thermo Fisher Scientific (1 mentions)
Pharm lyse buffer, by BD (1 mentions)
F4 80 pe, by Thermo Fisher Scientific (1 mentions)
Cd206 apc, by Thermo Fisher Scientific (1 mentions)
Cd45 fitc, by Thermo Fisher Scientific (1 mentions)
Cd8 apc, by Thermo Fisher Scientific (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Pd 1 pe, by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Roche (1 mentions)
Ifn γ pe cy7, by Thermo Fisher Scientific (1 mentions)
Kaluza software package, by Beckman Coulter (1 mentions)
Real time glo annexin 5 apoptosis and necrosis reagent, by Promega (1 mentions)
Cell f, by Olympus (1 mentions)
Annexin 5 apc, by Immunotools (1 mentions)
Enspire 2300 multimode plate reader, by PerkinElmer (1 mentions)
Ckx41 inverted microscope, by Olympus (1 mentions)
6 protocols using facslyric flow cytometry system
1
Fluorescent Probes for ROS and RNS Detection
2
Measuring GPR34 Mutant Expression by Flow Cytometry
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Expression of GPR34 mutants was measured by flow cytometry. In brief, HEK293A cells were transfected with human GPR34-encoding plasmid (250 ng), using polyethyleneimine. Cells were then suspended in 200 ml of D-PBS, containing 2 mM EDTA, and dispensed into 96-well V-bottom plates. After centrifugation for 1 min at 700 × g, the cells were suspended in 200 ml/well of FACS buffer (D-PBS, containing 0.5% BSA and 2 mM EDTA) and incubated for 30 min on ice. Cells were then centrifuged again for 1 min at 700 × g, resuspended in 25 ml/well of anti-human GPR34 serum (1/100 diluted), and incubated for 30 min on ice. After centrifugation for 1 min at 700 × g, cells were washed with D-PBS, resuspended in 25 ml/well of goat anti-mouse IgG conjugated with Alexa488 (Thermo Fisher Scientific, 10 mg/ml), and incubated for 15 min on ice. Cells were then centrifuged a final time for 1 min at 700 × g, washed with D-PBS, and resuspended in 150 ml/well of D-PBS, containing 2 mM EDTA. Flow cytometry analysis was performed with the BD FACSLyric Flow Cytometry System (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analysed by FlowJo Software (FlowJo, Ashland, OR, USA).
3
Immune profiling of tumor tissues
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Primary tumor tissues or spleens were dissociated using a gentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) after enzyme digestion in serum-free RPMI 1640 medium containing DNase I (1 U/mL; Roche, Basel, Switzerland) and collagenase D (1 mg/mL; Roche) for 20 min at 37 °C with gentle agitation. After single-cell dissociation, the cells were filtered through a 40 μm cell strainer, and the red blood cells were eliminated using BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, USA). The cells were stained by incubating at 4 °C for 1 h using the following antibodies: CD45-FITC, F4/80-PE, CD206-APC, CD86-PE/Cy7, CD8-APC, PD-1-PE, and IFN-γ-PE/Cy7 (e-Bioscience, CA, USA). After staining, the cells were analyzed using a BD FACSLyric flow cytometry system (BD Biosciences) and FlowJo software (Treestar, San Carlos, CA, USA).
4
PBMC Immunophenotyping by Flow Cytometry
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The isolated PBMCs were analysed on a BD FACSLyric Flow Cytometry System (BD Biosciences, Franklin Lakes, NJ, USA) with BD Multitest TM 6‐Colour TBNK, according to the manufacturer's instructions.
5
Multiparametric Analysis of Cultured Megakaryocytes
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Differentiation parameters of cultured megakaryocytes were analyzed by flow cytometry. Samples were stained with CD61-FITC and CD42b-PE antibodies (Abcam, Cambridge, UK). For megakaryocyte ploidy, cells were fixed overnight in ice-cold 70% ethanol at −20 °C. Samples were then incubated in 100 μg/mL of RNAse and propidium iodide solution and stained with antihuman CD61-FITC antibody. All samples were acquired with a BD FACSLyric™ Flow Cytometry System. Offline data analysis was performed using the Beckman Coulter Kaluza software package (Brea, CA, USA).
6
Real-Time Apoptosis and Necrosis Assay
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For real-time investigation of apoptosis and necrosis, cells were seeded at densities of 1 × 104 cells in 50 µl media into white 96-well plates and incubated for 24 hours at 37°C. Roginolisib (100 µM), paclitaxel (400 nM) or DMSO (0.1%) were added followed by immediate addition of Real Time-Glo Annexin V Apoptosis and Necrosis reagent (part# JA1011, Promega, Madison, WI, USA). Luminescence and fluorescence signals were monitored overtime up to 40 h using the EnSpire Multimode Plate Reader 2300 (PerkinElmer). For end-point investigation of apoptosis and necrosis, cells were seeded at densities of 1 × 105 cells in 1 ml media into 24-well plates and incubated for 24 hours at 37°C. Then, cells were treated with roginolisib (100 µM) or DMSO (0.1%) and incubated for a further 31 hours. After harvesting using accutase, cells were stained using Annexin V-APC (Immunotools, Friesoythe, Germany) and 25 µg/ml PI (propidium iodide) for 10 min. Cells were analysed by a BD FACSLyric Flow Cytometry System (BD, Heidelberg, Germany). Phase-contrast microscopy was used to investigate morphological changes. Cells (1 × 105 cells/2ml) were seeded into 6-well plates and incubated with roginolisib (100 µM) or DMSO for 24 h. Cells were imaged using a CKX41 inverted microscope and Olympus CellF (Olympus Life Science, Tokyo, Japan).
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