The mean fluorescence intensity of M1 or M2 phenotype cells was determined by evaluating the level of their corresponding markers: CD86 and CD206, respectively, via flow cytometry assay. After treatment in the designed study groups, the BV-2 cells were collected and suspended in phosphate-buffered saline (PBS). Subsequently, the BV-2 cells were stained with the fluorescence-labeled CD86 (R-PE, BD Pharmingen, Franklin Lakes, NJ, USA, 09275B, 1:100) and CD206 (Alexa Fluor-647, BD Pharmingen, Franklin Lakes, NJ, USA, 565250, 1:200) antibodies for 30 min in the dark at 4 °C. FACS Canto II (Beckman Coulter, Indianapolis, IN, USA) was applied for analyzing the expression of CD86 and CD206.
Facs canto 2
Manufactured by Beckman Coulter
Sourced in United States
The FACS Canto II is a flow cytometry instrument designed for advanced multiparameter analysis. It incorporates a three-laser configuration and can detect up to 10 fluorescent parameters simultaneously. The system is capable of high-speed data acquisition and analysis for a wide range of applications in research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by BD (1 mentions)
Cd8 fitc, by BD (1 mentions)
Il 2 pe, by BD (1 mentions)
Cd4 apc, by Thermo Fisher Scientific (1 mentions)
Ifn γ percp cy5, by BioLegend (1 mentions)
Cd3 apc efluor780, by Thermo Fisher Scientific (1 mentions)
Cd8 pe cy7, by BioLegend (1 mentions)
Diva software, by Beckman Coulter (1 mentions)
Trustain fcx antibody, by BioLegend (1 mentions)
Bn 2 system, by Siemens (1 mentions)
Immulite 1000 immunoassay system, by Siemens (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
6 protocols using facs canto 2
1
Evaluating M1 and M2 Macrophage Markers
2
Cytokine Profiling of Ag85A and ESAT-6 Responses
3
Quantifying PD-L1 Secretion in CHO Cells
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CHO cells expressing either human PD-L1 or human CD80 fused to miRFP670 (table S3) were plated at 100,000 cells per well into a U-bottom 96-well plate and centrifuged at 300g for 5 min, and the supernatant was discarded. Two hundred–microliter supernatant containing secreted PD-L1 nanobody was added to CHO cell lines either neat or diluted in 1:10 and 1:100 in FACS buffer. Cells were incubated at 4°C for 30 min before being washed in FACS buffer for analysis on a Beckman Coulter FACSCanto II (Fig. 6F ).
4
Macrophage Surface Marker Analysis
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Macrophages were incubated with TruStain FcX antibody (BioLegend, San Diego, CA, USA) for 10 min on ice to prevent unspecific binding of antibodies to Fc receptors. Then, cells were stained with rat anti-mouse CD86-APC (clone GL1, BioLegend, San Diego, CA, USA) or rat anti-mouse CD206-Brilliant Violet 421 (clone V068C2, BioLegend, San Diego, CA, USA) for 20 min at 4 °C in the dark and further washed with PBS supplemented with 2% FCS and 1 mM EDTA. Unstained cells were used as negative control for fluorescence. All data were collected by flow cytometry using FACSCanto II and DIVA software (Beckman Coulter, Brea, CA, USA).
5
Cytokine and Immunoglobulin Profiling in Chemotherapy
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Prior to treatment, and 2 weeks after the initial induction of chemotherapy, the serum levels of the cytokines IL-8, IL-6, IL-10, IL-1β, and sIL-2R were measured using the IMMULITE-1000 Immunoassay System (Siemens Healthineers, Erlangen, Germany) while the immunoglobulins IgA, IgG, IgM and IgE were detected by the BN II system (Siemens Healthineers). Lymphocyte subsets in the peripheral blood, including T, B, natural killer, cytokine-induced killer, T helper (Th), suppressor T (Ts), Th/Ts, and regulatory T cells (Treg) were tested using a FACSCanto™ II flow cytometer (Beckman Coulter, Inc., Brea, CA, USA), using a BD Multitest™ 6‐color TBNK kit and DIVA software.
6
Quantifying Cellular ROS Levels
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Alterations in the ROS levels were determined by dihydroethidium (DHE) staining (Molecular Probes, Eugene, OR, USA), which exhibits blue-fluorescence in the cytosol and can be oxidized by ROS in cells to form ethidium oxide, which can be incorporated into chromosomal DNA to produce red-fluorescent. Briefly, the cells were incubated with 4 μM DHE for 15 min at 37 °C. After washing twice with PBS, the population of ethidium bromide-stained cells was measured using FACS Canto II (EPICS XL, Beckman-Coulter, Miami, FL, USA and FACS Canto II, BD Biosciences, Mississauga, ON, USA) as the fraction of cells producing intracellular ROS, according to the manufacturer's protocol.
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