PBMCs were isolated from peripheral blood by density dependent centrifugation with Lymphoprep (Axis Shield, Norway). Total B cells were isolated by negative selection with a memory B cell isolation kit according manufacturer’s instructions (Miltenyi Biotech, USA). B cells were cultured as previously described (Cocco et al., 2012 (link)) using gamma-irradiated CD40L-L cells and 2 µg/ml F(ab′)two goat anti-human IgM/IgG/IgA (Jackson ImmunoResearch) supplemented with 20 U/ml IL-2 (Roche) and 50 ng/ml IL-21 (Peprotech) as the initial T-depedent stimulus. Cells were monitored for changes in cell surface antigen expression using anti-CD38-APC-Cy7 (HB-7; BD Biosciences), anti-CD138-APC (44F9; Miltenyi Biotec), as well as 7-AAD-PerCP-Cy5 (BD Biosciences) to identify live cells. Flow cytometry was performed using a or a CytoFLEX S or LX (Beckman Coulter). Analysis was performed using FlowJo v10 (TreeStar). Supernatants were collected at day 6 and day 13 from the differentiation assays and assessed for Ig levels using human IgM ELISA Quantitation Set (E80-100) or Human IgG ELISA Quantitation Set (E80-104) (Bethyl Laboratories Inc, USA) according to manufacturer’s instructions on a Berthold 96-well plate reader. ELISA absorbance values were analyzed at 450 nm and Ig concentrations calculated from standard curves.
7aad percp cy 5
Manufactured by BD
Sourced in United States
7AAD Percp Cy 5.5 is a fluorescent dye used in flow cytometry applications to detect and quantify cell death or apoptosis. It binds to DNA and emits a signal in the PerCP-Cy5.5 channel, allowing for the identification of non-viable cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex s or lx, by Beckman Coulter (1 mentions)
Human igg elisa quantitation set e80 104, by Fortis Life Sciences (1 mentions)
Memory b cell isolation kit, by Miltenyi Biotec (1 mentions)
Interleukin 2 (il 2), by Roche (1 mentions)
Il 21, by Thermo Fisher Scientific (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Cd49f pe, by BD (1 mentions)
Cd34 apc, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cd90 fitc, by BD (1 mentions)
Cd45ra pe cy7, by BD (1 mentions)
Cd4 pe cy7, by Thermo Fisher Scientific (1 mentions)
Ze5 cell analyzer, by Bio-Rad (1 mentions)
Cd45 apc efluor780, by Thermo Fisher Scientific (1 mentions)
Ly6c v450, by BD (1 mentions)
F4 80 bv510, by BD (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Ly6g pe, by BD (1 mentions)
Facsverse, by BD (1 mentions)
6 protocols using 7aad percp cy 5
1
Differentiation of Memory B Cells
2
Characterization of Cytokine-Expanded Cells
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PCs or cultured cells expanded with cytokines in the presence or absence of VPA were stained with the anti‐human antibodies 7AAD‐PerCP‐Cy5, CD34‐APC, CD90‐FITC, CD49f‐PE, and CD45RA‐PE‐Cy7 or their isotype‐matched controls (BD Biosciences, San Jose, California) for 20 minutes in room temperature, washed and analyzed using the FACS‐Canto II (BD Biosciences). Analyses were performed with BD FACSDiva Software and FlowJo Software (BD Biosciences).
3
Multicolor Flow Cytometry of Immune Cells
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For Cox8-mCherry-eGFP, Bio-Rad Ze5 Cell Analyzer was used. Cells were first sorted for mCherry positivity followed by eGFP. Analysis was done using FlowJo software. Flow cytometry analysis of immune cells was done using the Beckman Coulter MoFlo Astrios; immune cells from the colon were isolated by 25 mM EDTA digestion to remove epithelial cells, followed by a 0.5 mg/mL collagenase IV digestion, and were enriched for using a 40%–70% percoll gradient. Immune cells were stained for with CD45 APC eFluor 780, 1:200 (Invitrogen 47-0451-82); CD4 PECy7, 1:300 (eBioscience, 25-0041-82); CDllc FITC, 1:200 (BioLegend, 117305); CDllb APC, 1:250 (eBioscience, 17-0112-83); Ly6C V450, 1:300 (BD Biosciences, 560594), Ly6G PE, 1:300 (BD Biosciences, 560594), F4/80 BV510, 1:100 (BD Biosciences, 563633), 7AAD Percp Cy 5.5, 1:300 (BD Biosciences, 559925).
4
Apoptosis Measurement in Oxidative Stress
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Cells were washed with PBS after transfection for 48 h (overexpression group and negative control group with or without 200 umol/L H2O2 and N-Acetyl-l -cysteine (NAC) treatment for 24 h to induce or anti-apoptosis) and were then harvested using 0.25% trypsin (all supernatant liquid was collected). After centrifugation (1500 rpm) for 15 min, the supernatant liquid was discarded, and the precipitation was resuspended in 1 mL of binding buffer for another centrifugation. Then, the cells were resuspended in 250 ul of binding buffer, and 5 ul each of annexin V-FITC and 7-AAD-Percp-Cy5.5 (BD Biosciences, San Jose, CA, USA) were added. The cells were analyzed by a FACSVerse (Becton–Dickinson, FranklinLakes, NJ, USA) after incubation in the dark for 15 min.
5
Isolation and Characterization of iNKT Cells
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iNKT cells were sorted on FACSAria III (BD Biosciences). Murine antibodies used were CD19‐PerCP‐Cy5.5 (eBio1D3), βTCR‐FITC (H57‐597), CD4‐APC‐eFluor780 (RM4‐5), CD8α‐e450 (53‐6.7), IL4‐FITC (11B11), IFNγ‐FITC (XMG1.2), MHC‐II‐FITC (114.15.2), F4/80‐PE (BM8), CD170‐APC (IRNM44N), CD11C‐PE‐Cy7 (N4/8) and Ly6G‐PE Texas red (all from Thermo Fisher scientific); CD11b‐APC‐Cy7 (M1/70), Ly6C‐BV510 (HK1.4), CD45‐BV605 (30‐F11), and CD1d‐e450 (1B1) (all from BD). Human antibodies used were TCRVβ11‐FITC (C21) and 7‐AAD‐PerCP‐Cy5.5 (all from BD). iNKT cells were stained at 4°C for 30 min using α‐GalCer‐loaded CD1d tetramers provided by NIH Tetramer Core Facility, USA, followed by antibody staining. iNKT cells are defined as iNKT (TCRβ+CD1d‐αGC+); for Annexin V, staining set (Thermo Scientific) was used according to the manufacturer's instructions. For intracellular proteins, the FoxP3/transcription factor staining buffer set (eBioscience) was used according to the manufacturers’ recommendations.
6
Analyzing Dendritic Cell Maturation and Viability
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For maturation, DCs (2x106 cells/ml) were stimulated as for ELISA and supernatants were discarded. Next, cells were washed twice in PBSAz, incubated with anti-mouse CD16/CD32 BD Fc Block (3 μg/ml; BD Biosciences, San Jose, CA, 553141) for 10 min on ice to block unspecific binding. Subsequently the cells were stained with monoclonal anti-mouse CD40-PE (2μg/ml, 12–0401), CD86(B7-2)-APC (1.25 μg/ml,17–0862) (both from eBiosciences) and CD80(B7-1)-FITC (1.25 μg/ml; BD Pharmingen, San Diego, CA, 553768) for 45 min at RT. Cells were then washed twice in PBSAz, fixed and analyzed by flow cytometry. MFI was used as a measure of maturation based on counting 10,000 cells. To analyze cell viability, the DCs were washed twice in ice-cold DPBS after 1 or 20 h of stimulation with bacteria and their supernatant. Next, the cells were re-suspended in BD Binding buffer, stained with anti-mouse Annexin V-PE and 7-AAD-PerCpCy5.5 (BD Pharmingen, San Diego, CA, 559925) for 15 min at RT and analyzed by flow cytometry based on counting 5,000 cells. Percentages of apoptotic, dead or double positive cells were used. Data analysis was performed using FlowJo Version 10 software (Treestar Inc).
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