Single-cell suspensions of peripheral blood or these prepared from lung tissue were stained with the following directly labeled antibodies: PerCP-Cy5.5-anti-mouse CD11b (Clone M1/70, BD Pharmingen, 561114, 1: 100); APC-anti-mouse Gr1(Clone RB6-8C5, BD Pharmingen, 553129, 1: 100); BV421-anti-mouse Ly6G (Clone 1A8, Biolegend 127628, 1: 100); FITC-anti-mouse Ly6G (Clone 1A8, eBioscience, 11-9668-82, 1:100); PE- anti-mouse Ly6C (Clone HK1.4, Biolegend, 128008, 1:100); APC-anti-mouse CD8 (Clone 53-6.7, Biolegend, 100712, 1:100); BV421-anti-mouse CD4 (Clone GK1.5, Biolegend, 100438, 1:100); APC-anti-mouse IFNγ (Clone XMG1.2, Biolegend, 505810, 1:100); PE-anti-mouse IL-17A (Clone ebio17B7, Biolegend, 559502). Detailed antibody information was summarized in Supplementary Table 3 . Stained cells were collected and analyzed by FACSVerse device (BD Biosciences) and data was analyzed by Flowjo v10.
Facsverse device
Manufactured by BD
The FACSVerse is a flow cytometry device designed for the analysis of cells and particles. It is capable of rapidly measuring and analyzing multiple physical and chemical characteristics of single particles, including their size, granularity, and fluorescence properties. The FACSVerse is a versatile tool used in various fields, such as immunology, cell biology, and clinical diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
B cell isolation kit 2, by Miltenyi Biotec (2 mentions)
Percp cy5.5 anti mouse cd11b, by BD (1 mentions)
Pe anti mouse ly 6c, by BioLegend (1 mentions)
Apc anti mouse ifn γ, by BioLegend (1 mentions)
Bv421 anti mouse cd4, by BioLegend (1 mentions)
Pe anti mouse il 17a, by BioLegend (1 mentions)
Apc anti mouse cd8, by BioLegend (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Human t cell isolation kit, by STEMCELL (1 mentions)
Bz x700 all in one fluorescence microscope, by Keyence (1 mentions)
96 well flat bottom plate, by Corning (1 mentions)
5 protocols using facsverse device
1
Immune Cell Profiling by Flow Cytometry
2
Phagocytosis of E. coli by Macrophages
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The mice were anesthetized and their peritoneal lavage fluid was collected with 10 mL of PBS containing 0.1% BSA (0.1% BSA/PBS). Peritoneal exudate cells (2 × 10 5 /100 μL) were incubated with 100 μL of 0.2 mg/mL pHrodo Escherichia coli BioParticles conjugate for phagocytosis (REF:P35361; Life Technologies, Carlsbad, Calif) for 30 min at 37°C. After a 10-min incubation with Fc-blocker, the cells were stained to identify F4/80 + macrophages and the expression of intracellular PE-labeled E. coli was quantified by flow cytometry using a FACS Verse device (BD Biosciences, San Jose, Calif).
3
Quantifying MDSC Suppression of T Cell Activation
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Healthy donor CD3 + T cells were isolated using the Human T-Cell Isolation Kit (STEMCELL Technologies) and stained with CellTrace Violet (Thermo Fisher Scientific). The total MDSC and each MDSC subset that had been obtained by sorting healthy donor sample with a FACSAria III were cocultured with the stained CD3 + T cells (1 × 10 5 cells) at 1:1, 0.5:1, 0.33:1, 0.2:1, and 0.1:1 ratios in a 96-well flat-bottom plate (Corning). Dynabeads Human T-Activator CD3/CD28 (Invitrogen) were then immediately added to the CD3 + T cells at a 1:1 ratio to induce T cell activation. After 3 days of coculture at 37°C and 5% CO 2 , the CD3 + T cells were harvested and analyzed by flow cytometry using a FACSVerse device (BD Biosciences). CD3 + T cell division was measured by Violet dilution. In addition, CD3 + T cell cluster formations under the previously stated experimental conditions were evaluated using a BZ-X700 All-in-one Fluorescence Microscope (KEYENCE).
The interferon-gamma (IFN-γ concentrations in the supernatants of the 3-day cocultures were determined with a Human ELISA Kit according to the manufacturer's protocol (RandD SYSTEMS).
The interferon-gamma (IFN-γ concentrations in the supernatants of the 3-day cocultures were determined with a Human ELISA Kit according to the manufacturer's protocol (RandD SYSTEMS).
4
Humanized anti-CD40 IgG1-LALA Antibody Stimulation of B-cells
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Example 10
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- a. The stimulation of costimulatory receptors by humanized anti-CD40 IgG1-LALA antibodies was also tested on B-cells. PBMCs from three different donors were isolated from human buffy coat by Ficoll density gradient centrifugation and untouched B-cells were purified by negative magnetic enrichment using a B-cell isolation kit II (Miltenyi Biotech) according to manufacturer's instructions. 2×105 B-cells in 100 μl RPMI-1640+10% Human AB Serum were stimulated with antibodies concentration ranging from 500 to 0.2 ng/ml for 48 h. Stimulated B-cells were harvested, stained using fluorophore-labelled antibodies against HLA-DR, CD86 and CD80 (all from Miltenyi Biotech) and analyzed by flow cytometry on a BD FACSVerse device.
FIG. 15 displays the dose dependent stimulation of receptor expression in B-cells of one donor as fold of induction over isotype antibody control treatment. Fitting curves and EC50 calculation were obtained by using Excel (Microsoft) and XLfit (IDBS). The results demonstrate that the antibodies of the invention also stimulate costimulatory receptors on B-cells, however, the level of upregulation is lower than that observed in DCs.
- a. The stimulation of costimulatory receptors by humanized anti-CD40 IgG1-LALA antibodies was also tested on B-cells. PBMCs from three different donors were isolated from human buffy coat by Ficoll density gradient centrifugation and untouched B-cells were purified by negative magnetic enrichment using a B-cell isolation kit II (Miltenyi Biotech) according to manufacturer's instructions. 2×105 B-cells in 100 μl RPMI-1640+10% Human AB Serum were stimulated with antibodies concentration ranging from 500 to 0.2 ng/ml for 48 h. Stimulated B-cells were harvested, stained using fluorophore-labelled antibodies against HLA-DR, CD86 and CD80 (all from Miltenyi Biotech) and analyzed by flow cytometry on a BD FACSVerse device.
5
Human B-cell Activation Assay
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PBMCs from three different anonymous donors were isolated from human buffy coats by Ficoll density gradient centrifugation, and untouched B-cells were purified by negative magnetic enrichment using a B-cell isolation kit II (Miltenyi Biotec) according to the manufacturer’s instructions. 2 × 105 B-cells in 100 μL RPMI-1640 + 10% Human AB Serum were stimulated with CD40 antibody concentrations ranging from 500 to 0.2 ng/mL for 48 h. Stimulated B-cells were harvested, stained using fluorophore-labeled antibodies against CD86 and CD80 and analyzed by flow cytometry on a BD FACSVerse device. Fitting curves were calculated using GraphPad Prism.
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