Cells were stained for flow cytometry following standardized protocols, in brief, surface staining was performed at 4°C in staining buffer (DPBS/1% BSA/0.02% NaN3/10% FcR-block, Miltenyi) for 60 minutes using the directly-conjugated antibodies listed in Table 2 . In all cases, saturating antibody concentrations were used and dead cells were excluded by 7-AAD staining (BD Biosciences). Data were captured with a Calibur cytometer (BD Biosciences) and analyzed with FlowJo (Tree Star Inc).
Calibur cytometer
Manufactured by BD
Sourced in United States
The Calibur cytometer is a flow cytometry instrument used for the analysis and measurement of cells and particles in a fluid sample. It is designed to detect and quantify various cellular properties, such as size, granularity, and the presence of specific markers on the cell surface or within the cell. The Calibur cytometer provides accurate and reliable data for a wide range of applications in fields such as immunology, hematology, and cell biology.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (6 mentions)
Lsrfortessa x 20, by BD (3 mentions)
Primary antibodies for serinc5 and cell viability dye zombie uv, by BioLegend (2 mentions)
Cytoperm buffer, by BioLegend (2 mentions)
Anti ha 11 epitope tag pe 16b12, by BioLegend (2 mentions)
7 aad staining, by BD (1 mentions)
Fcr block, by Miltenyi Biotec (1 mentions)
Phycoerythrin conjugated rat igg isotype, by BD (1 mentions)
Fluorescein conjugated anti cd4 antibody, by BD (1 mentions)
Phycoerythrin conjugated anti cxcr3 antibody clone 173, by BD (1 mentions)
Collagenase type d, by Roche (1 mentions)
Cd138 pe, by BD (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Cell proliferation dye efluor 450, by Thermo Fisher Scientific (1 mentions)
Apc goat anti rabbit igg, by BD (1 mentions)
Annexin 5 fitc and pi, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis kit, by Keygen Biotech (1 mentions)
14 protocols using calibur cytometer
1
Flow Cytometry Surface Staining Protocol
2
Quantifying Conjunctival CXCR3+ T Cells
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Flow cytometry was performed to quantify the number of CD4+C-X-C chemokine receptor type 3 positive (CXCR3+) T cells in the conjunctiva as previously described [31 (link)]. The conjunctival tissues (four eyes per group) were harvested, dipped in phosphate-buffered saline (PBS), teased apart using scissors, and then shaken at 37°C for 60 minutes in the presence of 0.5 mg/mL collagenase type D (Roche Applied Science, Indianapolis, IN, USA). After incubation, the tissues were disrupted by grinding using a syringe plunger and passed through a cell strainer with a pore size of 100 mm. The cells were centrifuged at 1500 rpm for 7 minutes and then resuspended in PBS with 1% bovine serum albumin (BSA). After washing, the samples were incubated with the fluorescein-conjugated anti-CD4 antibody (BD Biosciences, San Jose, CA, USA), phycoerythrin-conjugated anti-CXCR3 antibody (clone 173, BD Biosciences), and isotype control antibody at 37°C for 30 minutes. The phycoerythrin-conjugated rat IgG isotype (BD Biosciences) was used as the control. The number of CD4+CXCR3+ T cells was counted using a fluorescence-activated cell sorting Calibur cytometer with the CellQuest software (both from BD Bioscience, Fullerton, CA, USA).
3
Flow Cytometric B-cell Analysis in PBMC and CSF
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Flow cytometric B-cell analysis was performed as described before.7 (link) Briefly, 1 × 106 PBMCs and, depending on cell count and volume of CSF samples, 3.0 × 103 to 5.7 × 104 (mean 1.65 × 104) CSF cells were stained with monoclonal antibodies specific for human B-cell markers (CD20-PerCP, CD27-PE, immunoglobulin D–fluorescein isothiocyanate [FITC], CD38-APC, human leukocyte antigen–DR-FITC, CD138-PE [BD Biosciences, Heidelberg, Germany]) and analyzed with a fluorescence-activated cell sorting Calibur cytometer and CellQuest software (BD Biosciences). Gating procedures are illustrated in figure e-1 at Neurology.org/nn .
4
Surface and Intracellular Staining of SERINC5
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HEK293T cells were washed and surface stained at 4 °C with primary antibodies for SERINC5 and cell viability dye Zombie UV (Biolegend). For intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with CytoPerm buffer (Biolegend). Primary antibody anti-HA.11 epitope tag-PE (16B12, Biolegend) was used to detect SERINC5 and IFITMs. Data were acquired on a BD LSR Fortessa X-20 or Calibur cytometer and analyzed using FlowJo.
5
Lymphocyte Subset Analysis in EAE Mice
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To assess the distribution of lymphocyte subsets in EAE mice, lymphocytes in the spleen were analyzed by flow cytometry. Spleens were collected from the mice from each group 18 days after immunization, and the cells isolated from the spleens were hemolyzed with red blood cell lysis buffer (Sigma). Then, the cell suspensions were adjusted to 1 × 107/mL; these were cultured in complete RPMI 1640 medium (Thermo Fisher Scientific, Suzhou, China) and then re-stimulated with a cell stimulation mixture for 4.5 hours before being collected and washed with phosphate buffered saline. Antibodies conjugated to fluorochromes were used to stain the Treg (CD4-PerCP-Cy5.5, Cat#561115; CD25-APC, Cat# 557192; and FoxP3-PE, Cat# 560408), T helper 1 (Th1; CD4-PE, Cat# 557308; CD3-APC, Cat# 553066; and IFN-γ-PerCP-Cy5.5, Cat# 560660), Th2 (CD4-PerCP-Cy5.5, Cat# 561115 and IL-4-APC, Cat# 554436), Th17 (CD4-PerCP-Cy5.5, Cat# 561115 and IL17-PE, Cat# 559502) and B (CD8-APC, Cat# 553035; CD3-PerCP-Cy5.5, Cat# 551163; and CD19-PE, Cat# 557399) cells, by following the manufacturer’s instructions. All antibodies were purchased from BD Biosciences (Heidelberg, Germany). Data were acquired from the fluorescence-activated cell sorting Calibur cytometer and the CellQuest software (BD) was used for analysis.
6
Mitochondrial Mass Measurement in Adipocytes
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The methodology was based on the protocol used by Shen et al. [25 (link)]. A fluorescent probe (Mito-Tracker Green FM; Molecular Probes, Eugene, OR, USA) was used to determine the mitochondrial mass of adipocytes [27 (link)]. Mature adipocytes (9 days of differentiation) were treated with palmitic or palmitoleic acid, trypsinised and centrifuged at 1500×g, 4 °C, for 5 min, resuspended in KRH buffer containing 0.1% BSA (w/v) and then incubated with 0.1 μmol/L MitoTracker Green FM in KRH buffer for 30 min at 37 °C. Cells were centrifuged at 1500×g, 4 °C, for 5 min and resuspended in 400 μL of fresh KRH buffer. Fluorescence measurements were carried out in CALIBUR cytometer (BD) in the FL1 channel. Ten thousand events were analyzed per experiment. Data were analyzed using Cell Quest software.
7
Surface and Intracellular Staining of SERINC5
8
Cell-cell Spread of HIV-1 in T Cells
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T cells were infected with 50,000 TCID50/million cells with VSV-G pseudotyped HIV-1 by gravity infection for 4 hours. For cell-cell spread assays HIV-1 infected T cells (donors) were analysed for Gag expression by flow cytometry 48h post-infection (p.i.). Uninfected T cells (targets) were labelled with 2.5 μM Cell Proliferation Dye eFluor 450 (Thermo Fisher Scientific)
according to manufacturer's instructions. HIV-1 infected Gag+ donors and dye-labelled targets were mixed in 1:1 ratio, incubated at 37°C for indicated time points and analysed by flow cytometry using a BD LSR Fortessa X-20 or Calibur cytometer. Data were analysed using FlowJo software.
according to manufacturer's instructions. HIV-1 infected Gag+ donors and dye-labelled targets were mixed in 1:1 ratio, incubated at 37°C for indicated time points and analysed by flow cytometry using a BD LSR Fortessa X-20 or Calibur cytometer. Data were analysed using FlowJo software.
9
FACS Analysis of Cell Populations
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The cells were analyzed on a fluorescence-activated cell sorting (FACS) Calibur cytometer using Cellquest software (Becton Dickinson) and FlowJo software (V. 7.6.4, TreeStar). The statistics presented are based on at least 10,000 events gated on the population of interest.
10
Murine Dendritic Cell Differentiation
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The method used by Inaba et al. was followed [16 ]. The tibiae and femurs were dissected from C57BL/6 6-weeks old female mice euthanized in a CO2 chamber. The bone marrow was flushed with a syringe filled with RPMI 1640 medium; a lysis buffer was used to deplete red cells. Cells were growth in RPMI-1640 with 5% heat-inactivated FBS, 20 μg/mL gentamicin, 100 mL 2-mercaptoethanol (0.1 M/L), and HEPES 1M (25 ml/L). To drive DCs differentiation, 20 ng/ml recombinant GM-CSF was added to the culture medium. After three days complete culture medium was added; on day 6, immature DCs growing in conglomerates were recovered and rinsed at 453 g for 5 min with PBS. By FACS, CD11c was measured to determine DC maturation (Calibur Cytometer (Beckton Dickinson, San Diego, CA, USA).
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