LN cell suspensions obtained after a mechanical dissociation were processed as described [24] and stained with the following mAbs from BD Biosciences: CD4 PercP or AlexaFluor 700, CD25 APC, CD44 PE-Cy7 or APC, and CD62L PE. Intracellular labeling of transcription factor Foxp3 by anti-Foxp3 Ab conjugated to Pacific Blue (FJK-16s, e-Bioscience, San Diego, CA, USA) was performed according to manufacturer's recommendations. Isotype-irrelevant mAbs were used as controls. Lymphocytes were acquired on an LSR-II TM Flow Cytometer and analyzed with FlowJo R (Tree Star) software. Cells were sorted from four individual mice on a FACS Aria TM cytometer with a purity over 90% as follows: CD4 + CD25 high CD44 high CD62L low activated Treg cells (amTreg); CD4 + CD25 high CD44 low CD62L high nTreg cells; CD4 + CD25 -(Teff). All available cells from each LN of each individual mouse were sorted. As previously shown [25] , over 90% of sorted amTreg cells and nTreg cells expressed Foxp3 and were bona fide Treg cells, while Teff cells did not express Foxp3.
Alexa fluor 700
Manufactured by BD
Sourced in United States
Alexa Fluor 700 is a fluorescent dye that can be used in various biological applications, such as flow cytometry and fluorescence microscopy. It has an excitation maximum at 702 nm and an emission maximum at 723 nm, making it suitable for detection in the far-red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm, by BD (4 mentions)
Brefeldin a, by Merck Group (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Apc cy7, by BD (2 mentions)
Cd38 fitc, by STEMCELL (2 mentions)
Golgistop, by BD (2 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
Cd62l pe, by BD (1 mentions)
Flowjor, by Tree Star (1 mentions)
Cd4 percp, by BD (1 mentions)
Cd44 pe cy7, by BD (1 mentions)
Cd25 apc, by BD (1 mentions)
Granzyme b, by BioLegend (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Anti α6 integrin conjugated to pe, by BD (1 mentions)
Anti cd19 apc, by BD (1 mentions)
Anti cd19 pe cy7, by BD (1 mentions)
17 protocols using alexa fluor 700
1
Identifying and Sorting Regulatory T Cells
2
Multiparametric Flow Cytometry Assay
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Restimulated splenocytes (106 cells/well) were treated with 10 µg/ml of brefeldin A (Sigma-Aldrich, St. Louis, Missouri, USA) at 37°C/5%CO2 4 h before staining. Spleen cells were stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit for 633 or 635 nm excitation (1:1,000, Invitrogen™, Waltham, Massachusetts, USA) followed by blocking of Fc receptors with CD16/CD32 Rat anti-Mouse (clone: 93, 1:500). For surface staining, antibodies to CD3e (clone: 145-2C11)-FITC, CD4 (clone: RM4-5)-PE, CD8a (clone: 53-6.7)-PerCP-Cyanine5.5, and CD69 (clone H1.2F3)-Alexa Fluor® 700 (BD Biosciences, Franklin Lakes, New Jersey, USA) were used. Afterward, cells were fixed and permeabilized using BD Cytofix/Cytoperm™ (BD BioSciences, Franklin Lakes, New Jersey, USA). Intracellular staining was performed with antibodies to IFN-γ (clone: XMG1.2)-APC and Granzyme B (clone: QA18A28, BioLegend®, San Diego, California, USA)-BV421. Cells were suspended in PBS and acquired using BD LSR Fortessa X-20 and BD FACSDiva (version 9.0, BD Biosciences, Franklin Lakes, New Jersey, USA). Data were analyzed with FlowJo™ (version 10.8.1, BD Biosciences, Franklin Lakes, New Jersey, USA). Antibodies were used at 1:200 dilution and were purchased from eBioscience™ (Invitrogen™ Waltham, Massachusetts, USA) unless otherwise stated.
3
Analysis of Bulge Hair Follicle Stem Cells
4
Cytotoxicity Assays for Cell Lines and Primary Material
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Killing of cell lines was tested in standard 51chromium release assays as described previously [9 (link)]. Killing of primary material was analyzed in FACS-based cytotoxicity assays. 50,000 target cells were co-cultured with T cells in effector/target (E/T) ratio 3:1 overnight. After overnight culture cells were stained with specific antibody panels and SYTOX Blue Dead Cell Stain (Invitrogen by Thermo Fisher Scientific) was added prior to acquisition to identify living cells. Samples were acquired by FACS using fixed flow rates and stable acquisition was validated using Flow-Count Fluorospheres (Beckman Coulter). Co-cultures with activated B cells were stained using anti-CD3 (Alexa Fluor 700, BD Pharmingen), anti-CD19 (PE-Cy7, BD Pharmingen), anti-IgA (PE, Miltenyi), anti-IgG (FITC, DAKO) and anti-IgM (APC, BD). For primary MM, 50,000 patient-derived BM mononuclear cells were used as target cells. Co-cultures were stained with anti-CD3, anti-CD19 (APC, BD Pharmingen), anti-CD45 (FITC, BD), anti-CD38 (PE-Texas Red, Invitrogen), and anti-CD56 (PE-Cy7, BD Pharmingen). MM cells were defined as CD3neg, CD19neg, CD45neg−int, CD38pos, CD56pos.
5
Detecting IL-4Rα Expression in Immune Cells
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IL-4Rα surface expression was detected on live cells isolated from the peritoneum or lamina propria by phycoerythrin (PE) anti-CD124 (IL-4Rα, M-1). Cell subpopulations were identified with Alexa Fluor 700, BD Horizon V450, APC, or PE-Cy7 for F4/80, Ly6G, CD11c, and CD11b (BD Pharmingen). Stained cells were then acquired on a LSRII flow cytometer (BD Bioscience), and data were analyzed using FlowJo software (TreeStar Inc.).
6
Plasmablast Frequency in Macaque Vaccines
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The frequency of plasmablasts (PBs) was measured in the blood of 12 macaques vaccinated with ALVAC-SHIV/gp120 Alum before vaccination and 7 days after the last immunization. Cells were stained with CD3 (Clone SP34-2), CD14 (Clone M5E2), CD16 (Clone 3G8), and CD56 (Clone B159), all conjugated in ALEXAFluor700 (BD Biosciences; San Jose, California, USA), PE-Cy5-CD19 (Clone J3-119, Beckman Coulter; Brea, California, USA), QDOT-650-CD20 (Clone 2H7, eBioscience; San Diego, California, USA), FITC-CD38 (Clone AT-1, StemCell; Massachusetts, USA), BV421-CD39 (Clone MOCP-21, BioLegend; San Diego, California, USA), PE-Ki67 (Clone B56, BD Biosciences), and anti-CXCR3 (CXCR3/CD183 PE-CF594 conjugated). Clone 1C6BD (Biosciences cat. #11718; anti-α4β7) was kindly provided by Dr. Ansari through the NIH AIDS Reagent Program, Division of AIDS, NIAID. Cells were permeabilized with Cytofix/Cytoperm (BD Biosciences). Acquisition was performed on an LSRII cytometer (BD Biosciences) and data were analyzed by FlowJo software (TreeStar; Ashland, Oregon, USA). Plasmablasts were gated as previously described: lineage-(CD3- / CD14- / CD16- / CD56+) / (CD19+/ CD20+ / (CD21-CD27+) / Ki67+/++ / (CD38+/++ / CD39+) [18 (link)]. The frequency of PBs expressing CXCR3 or α4β7 was then calculated.
7
Multiparametric T-cell Functionality Analysis
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Dead cells, B-cells, and monocytes were excluded using Aqua (L34957, Invitrogen), CD19 APC Alexafluor750 (1072337A, Invitrogen) and CD14 APC Alexafluor750 (773927B, Invitrogen) antibodies, respectively. T-cells were defined using CD3 (brilliant violet 570, B152103, Biolegend), CD8 (pacific blue, 22416, BD Bioscience) and CD4 (PE-Cy5.5, 1049514A, ebiosciences) surface staining antibodies. We defined CD4+ T-cells as the CD3+CD8−CD4+ T-cell phenotype, and CD8+ T-cells as the CD3+CD4−CD8+ T-cell phenotype. Virus-specific CD4+ and CD8+ T-cell responses were determined using intracellular staining for IFN-γ (Alexafluor 700, 21128, BD Biosciences); IL-2 (APC, 341116, BD Biosciences); Perforin (FITC B-D48 clone, F111124, Diaclone); and TNF-α (PE-Cy7, E07677-1632, ebiosciences). Simultaneous secretion of IFN-γ, IL2, Perforin and TNF-α in response to stimulation with the respective HIV-1 peptides was measured. We defined polyfunctionality as the concurrent secretion of three or more T-cell functions.
8
Detailed T-cell Phenotyping Protocol
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T-cell phenotyping was performed on fresh PBMNC or follow-up samples stored in liquid nitrogen. For the analysis of GrB+ T-cells, the cells were first stained for cell surface markers; α-CD45 APC-H7 (BD 641417), α-CD3 APC (BD 345767), α-CD4 PerCP (BD 345770), and α-CD8 PE-Cy7 (BD 335822), and then fixed and permeabilized with Fix/Perm (BD 554714), and intracellular GrB was stained (Alexa Fluor 700, BD 561016)). The memory cell subsets of CD4+ and CD8+ T-cells were studied by the following panel: α-CD45-APC-H7, α-CD3-PeCy7 (BD 557851), α-CD4-PerCP, α-CD45RA AlexaFluor700 (BD 560673) and α-CCR7-PE (R&D Systems FAB197P). 50´000 CD45+ cells were acquired with FACSAria (BD) and analyzed with FlowJo.
9
Murine Immune Cell Activation and Phenotyping
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All following conjugated Abs are from BD:APC conjugated anti–mouse CD25, -CD4, -CD45.1, -CD11c, -IL-12p70; Alexa Fluor 700–conjugated anti-CD3, -CD4, and -CD11c; PE-conjugated anti-CD3, -CD19, and -CD49b; FITC-conjugated anti-CD3, -CD19, -CD49b, and isotype control; biotin anti-CD4, -CD8, -DX5, -B220, -CD3, -CD11b, -Ly-6G, and -Ter119; and purified anti-CD16/CD32 (2.4G2). CD11c and streptavidin beads (SA) from Miltenyi Biotec; CFSE, live dead fixable aqua, CL075, and LPS from Invitrogen; ATRA from Sigma-Aldrich; hTGF-β1, anti–mouse TGF-β (1D11), anti-CTLA4, and Ig isotype control from R&D Systems.
10
PBMC Characterization by Flow Cytometry
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For each individual cell sample, 5×106 PBMCs were quickly thawed at 37°C and diluted in 40 ml cold wash buffer (PBS supplemented with 2.5% FBS (Life Technologies Ltd. Paisley, UK) and 0.1% sodium azide). The cell suspension was centrifuged at 250×g for 5 min and after discarding the supernatant, the pellet was resuspended in 400μl wash buffer. Each sample was stained for 1 h in a light-protected environment at 2°C with titrated amounts of anti CD19 labelled with Alexa Fluor 700 (BD Biosciences). Samples were analyzed using a BD LSR II Special Order System, controlled by the BD FACSDiva 6.0 software (BD Biosciences). A preliminary forward scatter (FSC) versus side scatter (SSC) gate was used to identify lymphocytes and, depending on sample size, a total of up to 100.000 in-gate events were recorded. All datasets were migrated to FlowJo 7.6.5 (Treestar Inc. Ashland, OR, USA) for further gating and analysis.
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