The peritoneal macrophages treated as in the section, ‘In vitro stimulation of peritoneal macrophages’ were harvested from culture plates using a 5 mM cold PBS-EDTA mixture for 10 min, washed, and then re-suspended in an FACS buffer (BD Biosciences). To avoid unspecific unions with antibodies, anti-mouse CD16/32 (93, dil. 2:100) was added for 20 min at 4°C to block the Fc receptors on the cell surface of macrophages. Then cells were washed with FACS buffer (BD Biosciences) and stained as indicated, with antibodies against F4/80 (BM8, 1:100), MHC-II (M5/114.15.2, 1:100), CD206 (C068C2, 2:100), IL-4Rα (I015F8, 4:100), PD-L1 (B7-H1, 2:100), PD-L2 (TY-25, 2:100), CD80 (16-10A1, 4:100), and CD86 (GL-1, 2:100) in staining buffer at 4°C and used according to manufacturer’s instructions (Biolegend). The stained cells were analyzed on a FACsCalibur flow cytometer (BD Biosciences) using Cell Quest and FlowJo software. Parallel samples of the cells were stained with the corresponding isotype control. T CD4+ cells obtained from the section, ‘Co-culture of peritoneal macrophages with lymphocyte T cells’ were washed with the FACS buffer (BD Biosciences), marked with anti-CD4 (GK1.5, 1:100), anti-CD25 (3C7, 1:100) and anti-CD69 (H1.2F3, 1:100) antibodies (Biolegend), and analyzed on Attune NxT flow cytometer (Thermo Fisher) using FlowJo software.
Facs buffer
Manufactured by BD
Sourced in United States, Germany, Spain
FACS buffer is a specialized solution used in flow cytometry applications. It is designed to maintain the integrity and viability of cells during sample preparation and analysis. The buffer helps to preserve the cellular structure and ensure accurate results in flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (9 mentions)
Facscalibur, by BD (6 mentions)
Fc blocker, by BD (3 mentions)
Macs buffer, by BD (3 mentions)
Fortessa flow cytometer, by BD (2 mentions)
Anti mouse cd16 32 antibody, by BioLegend (2 mentions)
Facs buffer, by BioLegend (2 mentions)
Cd80 fitc, by BD (2 mentions)
Cd86 pe, by BD (2 mentions)
Cd40 pe, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Cd14 percp, by BD (2 mentions)
Tlr2 fitc, by Thermo Fisher Scientific (2 mentions)
Tlr4 pe, by Thermo Fisher Scientific (2 mentions)
Cellquest pro software, by BD (2 mentions)
Canto 2 flow cytometer, by BD (2 mentions)
Fc block, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Perm wash buffer, by BD (2 mentions)
Cytofix, by BD (2 mentions)
63 protocols using facs buffer
1
Phenotypic Analysis of Peritoneal Macrophages
2
Pericyte Isolation and Flow Cytometry
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The second generation of primary pericytes in different groups was lysed with 0.25% trypsin without EDTA. Cells were collected and washed in ice-cold PBS twice. Cell suspensions were incubated for 30 min with anti-CD45 APC (Biolegend, USA) in FACS buffer (Becton Dickinson, USA), washed, and incubated with goat anti-rabbit IgG in FACS buffer for 30 min. The cells were then suspended in the same buffer for analysis. All data were collected and analyzed by FlowJo software (Treestar, Ashland, OR, USA).
3
Flow Cytometry Viability Staining Protocol
4
Isolation and Analysis of PBMCs
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In order to isolate peripheral blood mononuclear cells (PBMCs) from each sample, 1 mL of heparinized whole blood from CHB patients or healthy controls was collected and subjected to Ficoll-Hypaque density gradient centrifugation. After centrifugation, PBMCs were collected and washed with FACS buffer (phosphate-buffered saline [PBS] containing 0.5% BSA, BD Biosciences). For T-cell staining, the collected PBMCs were stained with the following fluorochrome-conjugated anti-human antibodies: CD3-APC, CD8-FITC, PD-1-PE, Tim-3-PE, CD3-APC, CD4-PerCP and CD25-FITC (Biolegend, San Diego, CA, USA) for 30 min. The stained cells were then washed with FACS buffer (BD Biosciences) and resuspended in a Fixation/Permeabilization Solution (BD Biosciences), prior to being washed and resuspended with PBS, and acquired on a FACSCalibur flow cytometer (BD Biosciences). After gating, the proportions of CD3+CD8+PD-1+, CD3+CD8+Tim-3+ and CD3+CD4+CD25high cells were determined. The data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
5
Necroptosis Detection in Adherent and Floating Cells
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Both adherent and floating cells were collected and transferred into 1.5 mL reaction tubes. The samples were washed twice with FACS buffer (BD Pharmingen, Heidelbery, Germany), centrifuged at 500× g and the supernatant were aspirated. Samples were then resuspended at a concentration of 1 × 106 cells/mL in FACS buffer (BD Pharmingen, Germany) and transferred (100 μL) into round-bottom 12 × 75 mm polystyrene Falcon tubes (Thermo Fisher). For necroptosis detection, cells were stained by mixing 5 μL of Annexin-V FITC and 5 μL propidium iodide (PI) in staining buffer. Following incubation at RT and protected from light for 15 min, 400 μL of staining buffer was finally added to every tube. Samples were analyzed using a FACS Vantage Flow Cytometer (Sony Biotechnology, Berlin, Germany).
6
Flow Cytometry Analysis of Ferumoxytol-Treated NK-92MI Cells
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NK-92MI cells were treated with ferumoxytol for 24 h, then washed with cold DPBS and FACS buffer (BD Bioscience). Cells resuspended in FACS buffer were blocked with Fc blocker (BD Bioscience) for 15 min at 4 °C, and then incubated with dye-conjugated antibodies for 1 h at 4 °C. Antibody-labeled samples were fixed with 4% paraformaldehyde. Antibodies used for flow cytometry were purchased from Biolegend. Flow cytometry analysis was performed using BD fortessa flow cytometer (BD Bioscience).
7
Characterization of Cells in Agarose Capsules
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Before transplantation, we analyzed the cells in the capsules around the agarose rods. After removing the agarose rods, the capsules were carefully excised, cut, and minced into small fragments. Subsequently, these fragments were passed through a 100-μm nylon mesh using a sterile syringe plunger and then centrifuged for 5 min at 1500 rpm at 4 °C. The red blood cell lysis buffer Hybri-Max (Sigma-Aldrich) was mixed with the resulting suspension. After washing with fetal bovine serum stain buffer (FACS Buffer; BD Biosciences, San Jose, CA), cells from which red blood cells were removed were first blocked with antimouse CD16/32 antibody (clone:93, BioLegend, San Diego, CA) for 20 min at 4 °C, followed by staining with LIVE/DEAD Fixable Dead Cell Stains (Thermo Fisher Scientific) to discriminate dead cells and then stained with antibodies for surface markers, namely CD11b (clone: M1/70, BV510, BioLegend), Ly6g (clone:1A8, PE, BioLegend), Ly6C (clone: AL-21, BD Biosciences), F4/80 (clone: BM8, PE-Cyanine7, Invitrogen), CD86 (clone: GL-1, BV421, BioLegend), and CD206 (clone: MR5D3, AF647, BD) for 20 min at 4 °C. After washing with FACS Buffer, the cells were resuspended and run on a BD FACSCanto II flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ashland, OR).
8
Phenotypic Analysis of Activated Macrophages
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THP1 differentiated macrophages were stimulated 24h with LPS 100 ng/ml for 24 hours harvested after trypsin digestion, centrifuged at 400g for 5 min, washed and resuspended with FACS buffer (BD Biosciences), and transferred to FACS tubes (BD Biosciences). Fixable Viability Dye eFluor 780 (eBioscience) was added at 1:1,000 in PBS and incubated for 20 min at 4°C. Cells were then washed with FACS buffer were stained with antibodies specific for either CD33 or CD38 (Biolegend), conjugated with Alexa Fluor 700. Staining was performed in a final volume of 100 μl with antibody dilution 1:100 for 30 min on ice. Cells were washed twice with FACS buffer and resuspended in a final volume of 300 μl, filtered through a 100-μm cell strainer and analysed using a FACS Fortessa (BD Biosciences). Live macrophages were gated based on viability dye. All data generated were analysed using FlowJo software (TreeStar).
9
Monocyte Surface Marker Expression by Flow Cytometry
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Cells were incubated with the following monoclonal antibodies: CD14-PerCP (BD Pharmingen, USA), CD16-FITC (BD Pharmingen), CD80-FITC (BD Pharmingen), CD86-PE (BD Pharmingen), CD40-PE (BD Pharmingen), TLR2-FITC (eBioscience, USA), and TLR4-PE (eBioscience). In isotype controls cells were stained with IgG1 conjugated with the respective fluorochromes (BD Pharmingen, eBioscience). After 30 min of incubation at 4°C, cells were washed twice in FACS buffer (Becton Dickinson, USA). The expression of monocytes surface markers was measured by flow cytometry (FACSCalibur, Becton Dickinson) and analyzed by Cell Quest software (Becton Dickinson). The results were based on analysis of at least 100,000 cells and were shown as the percentage of positively labeled cells. Moreover, the mean fluorescence intensity (MFI) values of gated monocytes positive for individual markers were determined.
10
Immunophenotyping of PBMC by Flow Cytometry
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The PBMC were stained with monoclonal antibodies and characterized by flow cytometry. In all patients, a Live/Dead fixable near-intensity ratio (IR) dead cell stain kit (Molecular Probes) was added to the cells before antibody staining, thus allowing the exclusion of dead cells from the analysis. Supplementary Table 5 shows the specificity, clone, fluorochrome, and source of the antibodies used. Cells were labeled in FACS buffer on ice and in the dark for 20 minutes following Fc block incubation (Becton Dickinson). Cells were further
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