On D0, PBMCs from 6 healthy women donors (from 25 to 35 years old) were incubated at the density of 200,000 cells/well in 96-well plates in Roswell Park Memorial Institute (RPMI) 1640 medium (Panbiotech, P04-17500), added with 2% human decomplemented serum, 1 mM non-essential amino acids, 1 mM pyruvate, 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, in the presence of HPV16(L1) peptide mix at the final dilution of 1/450 v/v. On D2, either the Veh., MIM-1; -2; -3; -4 or -5 were added to the medium and incubated for the next 72 h at the final sucrose–lactose concentration of 11 mM. Controls with either HPV16(L1) or IL-2 (20 ng/mL) were also run in parallel. On D5, the cells were harvested, saturated with FcBlock solution (BD, 564220), immune-stained with fluorescent anti-CD3, anti-CD4, anti-CD8, anti-CD71, anti-CD95, anti-CD28 and anti-HLA-DR antibodies (all purchased from BioLegend), fixed, and analyzed by flow cytometry. The discrimination of the cell sub-populations is as follows: CD4+: CD3high; CD4high; CD8low; SSClow, CD8+: CD3high, CD4low, CD8high, SSClow, CD4−/CD8−: CD3high; CD4low; CD8low; SSClow. The supernatants (SNs) were retrieved and the cytokine levels of IFN-γ, IL-6, and interferon- γ inducible protein (IP-10) were assessed by enzyme-linked immunosorbent assay (ELISA).
Anti cd95
Manufactured by BioLegend
Anti-CD95 is a monoclonal antibody that binds to the CD95 receptor, also known as Fas or APO-1. CD95 is a cell surface protein that plays a role in programmed cell death or apoptosis. The Anti-CD95 antibody can be used in flow cytometry and other immunoassays to detect and quantify CD95 expression on cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11c, by BioLegend (4 mentions)
Anti cd4, by BioLegend (3 mentions)
Lsrfortessa, by BD (3 mentions)
Anti cd45, by BioLegend (3 mentions)
Anti cd21, by BioLegend (3 mentions)
Anti cd19, by BD (3 mentions)
Anti igd, by BD (3 mentions)
Anti cd27, by BD (3 mentions)
Anti pd 1, by BioLegend (3 mentions)
Anti cd86, by BioLegend (3 mentions)
Anti cd8, by BioLegend (2 mentions)
Anti cd40, by BioLegend (2 mentions)
Anti f4 80, by BioLegend (2 mentions)
Anti b220, by BioLegend (2 mentions)
Anti gl7, by BioLegend (2 mentions)
Anti cxcr5, by BioLegend (2 mentions)
Live dead detection kit, by Thermo Fisher Scientific (2 mentions)
Anti hla dr antibody, by BioLegend (1 mentions)
Anti cd28, by BioLegend (1 mentions)
Fcblock solution, by BD (1 mentions)
8 protocols using anti cd95
1
HPV16 Peptide-Mediated Immune Response
2
Phenotyping of Human B Cell Subsets
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After thawing, PBMC (2 x 106/ml) were stained for 20 min at room temperature with the following antibodies: anti-CD45 (BioLegend 368540), anti-CD19 (BD 555415), anti-CD27 (BD 555441), and anti-IgD (BD 555778) to measure naive (IgD+CD27-), IgM memory (IgD+CD27+), switched memory (IgD-CD27+), and DN (IgD-CD27-) B cells. To measure membrane expression of markers associated with IA, B cells were also stained with anti-CD95 (BioLegend 305635), anti-CD21 (BioLegend 354911), anti-CD11c (BioLegend 301625), anti-CD86 (BioLegend 374215), anti-HLADR (BioLegend 307617), anti-PD1 (BioLegend 329907) antibodies. Up to 104 events in the B cell gate were acquired on an LSR-Fortessa (BD) and analyzed using FlowJo 10.0.6 software. Single color controls were included in every experiment for compensation. Isotype controls were also used in every experiment to set up the gates.
3
Comprehensive Immune Cell Profiling in Sepsis
4
Comprehensive Antibody Validation for Immunoblotting and Flow Cytometry
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Anti-mouse β-actin (AF0003; 1:1,000 dilution), Horseradish peroxidase (HRP)-anti-rabbit immunoglobulin G (IgG) (A0208; 1:3,000 dilution) and HRP-anti-mouse IgG (A0216; 1:3,000 dilution) were purchased from Beyotime Institute of Biotechnology (Haimen, China). Anti-ACK1 (PA5-102625) antibody was purchased from Invitrogen (Carlsbad, CA, USA). Anti-p-p65 (9246), anti-p65 (9242), anti-p-Erk (9101), anti-Erk (9107), anti-p-JNK (9251), anti-JNK (9251), anti-p-p38 (4511) and anti-p38 (9228) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). For flow cytometry purposes, fluorochrome-conjugated anti-B220 (Cat#: 103206), anti-CD4 (Cat#: 100406), anti-CD40 (Cat#: 124610), anti-F4/80 (Cat#: 123108), anti-CD86 (Cat#: 105012), anti-CD11c (Cat#: 117310), anti-GL7 (Cat#: 144608), anti-CD95 (Cat#: 152604), anti-CXCR5 (Cat#:145506), and anti-PD-1 (Cat#:135206) antibodies were purchased from BioLegend. Unless specifically listed otherwise, all flow cytometry antibodies were stained at a 1:100 dilution.
5
Comprehensive Immune Cell Profiling
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The cells were resuspended in ice-cold PBS containing 1% FBS and were stained with anti-CD3, anti-CD4, anti-CD8, anti-B220, anti-CD38, anti-CD138, anti-PD-1, anti-CXCR5, anti-CD62L, anti-OX40, anti-IgD, anti-CD95, anti-GL7, anti-CD11c, anti-CD80, anti-CD86, anti-MHC II, anti-CD40, anti-CD69, anti-CD11b, anti-Gr-1, and anti-F4/80 antibodies (Biolegend). Intracellular staining of IFN-γ, TNF-α, IL-17a, Helios, and Foxp3 was performed after 4 h of stimulation with PMA and ionomycin following a standard protocol (BD Biosciences). The results were obtained on a BD FACS Fortessa flow cytometer and were analyzed using FlowJo software.
6
Evaluating T-cell Activation and Apoptosis
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The surface expression of CD69 and CD25 markers of T-cell activation were measured using flow cytometry. Cells were stained with Zombie Aqua Fixable Viability Kit (Biolegend) and then stained with the following fluorescently-conjugated monoclonal antibodies: APC mouse anti-human CD3 (Clone UCHT1, Biolegend), V450 mouse anti-human CD4 (Clone RPA-T4, Biolegend), PE mouse anti-human CD69 (Clone FN50, Biolegend), PerCP-Cy5.5 mouse anti-human CD25 (Clone M-A251, Biolegend). Apoptosis was determined using Propidium iodide and Annexin V Pacific blue (Biolegend). anti-CD95 (1 ug/ml; clone E0S9.1; Biolegend) stimulation for 6 h was used as positive control for apoptosis. Cells were analyzed using LSR II flow cytometer and FACSDiva software. Data were analyzed with FlowJo software.
7
Identification of B Cell Subsets
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Cells from the SVF (5x105) were stained for 20 minutes at room temperature with a Live/Dead detection kit (InVitrogen 1878898), anti-CD45 (Biolegend 368540), anti-CD19 (BD 348794), anti-CD27 (BD 555441) and anti-IgD (BD 555778) antibodies. This protocol allows the identification of the major B cell subsets [naive (IgD+CD27-), IgM memory (IgD+CD27+), switched memory or swIg (IgD-CD27+), and double negative or DN (IgD-CD27-) B cells]. To measure membrane expression of markers associated with autoimmune B cells, cells were also stained with anti-CD95 (Biolegend 305635) and anti-CD21 (Biolegend 354911). Up to 105 events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FlowJo 10.5.3 software. Single color controls were included in every experiment for compensation. Isotype controls were also used in every experiment to set up the gates.
8
Comprehensive Phenotyping of B Cell Subsets
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PBMC (1x106 cells) or 5x105 cells from the Stromal Vascular Fraction (SVF) of the SAT were stained for 20 min at room temperature with the following antibodies: Live/Dead detection kit (InVitrogen 1878898), anti-CD45 (Biolegend 368540), anti-CD19 (BD 555415), anti-CD27 (BD 555441) and anti-IgD (BD 555778) to measure naive (IgD+CD27-), IgM memory (IgD+CD27+), switched memory (IgD-CD27+), and DN (IgD-CD27-) B cells. To measure membrane expression of markers associated with autoimmunity, B cells were also stained with anti-CD95 (Biolegend 305635), anti-CD21 (Biolegend 354911), anti-CD11c (Biolegend 301625) antibodies. To measure intracellular (ic) levels of phosphorylated STAT3, phosphorylated AMPK and total AMPK, cells were fixed and permeabilized with PBS containing 0.2% Tween and then stained for additional 20 min at room temperature with anti-phosho-STAT3 antibody (Tyr 705, #4113S), anti-phospho-AMPK antibody (Thr 172, #2535S) and anti-AMPK (#2532), respectively (all from Cell Signaling). Up to 105 events in the lymphocyte gate were acquired on an LSR-Fortessa (BD) and analyzed using FlowJo 10.0.6 software. Single color controls were included in every experiment for compensation. Isotype controls were also used in every experiment to set up the gates.
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