Blood samples were collected on Days 0 (pre-vaccination), 1, 3, 7, 14, 28 (post-vaccination), 35, 42, 56, 168 (post-vaccination), 182, 196 and 365. Venous whole blood from each individual was stimulated with either 10 µg/ml recombinant LEISH-F3, PBS (negative control), or 75 µg/ml PHA (positive control; Sigma-Aldrich, St. Louis, MO) for 12 hours at 37°C. Brefeldin A (10 µg/ml, Sigma-Aldrich) was added for the final 6 hours. Cells were treated with FACS Lysis Solution (BD Biosciences, San Jose, CA) and stained for surface CD3 (OKT3), CD4 (OKT4), CD8 (SK1), CD49d (9F10) and CD11a (HI111). Cells were permeabilized with eBioscience permeabilization buffer and stained for intracellular IFN-γ (4S.B3), TNF-α (MAb11), IL-2 (MQ1-17H12) and IL-10 (JES3-9D7). All antibodies were obtained from eBioscience. Samples were run on a BD LSR Fortessa (BD Biosciences) and data were analyzed with FlowJo software (Tree Star Inc, Ashland, OR). Supplemental Figures 1 and 2 depict the Day 0 and Day 182 staining controls.
Jes3 9d7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The JES3-9D7 is a laboratory equipment designed for general use in scientific research and analysis. It is a device that serves a core function, but no further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Facs lysis solution, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Mq1 17h12, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
3 protocols using jes3 9d7
1
Cytokine Response to LEISH-F3 Vaccine
2
Intracellular Cytokine Staining of PBMCs
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PBMCs were collected from patients, as described previously, and were incubated in a culture medium (RPMI 1640 + 10% fetal calf serum) and 1000× brefeldin A (Thermo Fisher Scientific) for 6 hours. Extracellular staining was performed before fixation and permeabilization (intracellular fix/intracellular permeabilization buffer, eBioscience). Intracellular interleukins were stained with anti–human IL-6 (hIL-6) (AS12, BD Biosciences) and anti–IL-10 antibodies (JES3-9D7, eBioscience).
3
Multicolor Flow Cytometry Immunophenotyping
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Peripheral blood cells were immunostained in the dark for 30 min at room temperature with TriColor-labelled [TC-phycoerythrin (PE)-cyanin 5 (Cy5)] monoclonal antibodies (mAbs) (Caltag, Burlingame, CA, USA), including anti-CD4 (clone S3.5), anti-CD8 (clone M-L233), anti-CD14 (clone TüK4), anti-CD16 (clone 3G8), and anti-CD19 (clone 4G7) mAbs. After lysis/fixation, the leukocytes were permeabilized by incubation with phosphate-buffered saline (PBS) permeabilization reagent (PBS supplemented with 0.5% [w/v] saponin [Sigma, St Louis, MO, USA]) for 10 min at room temperature. Fixed/Permeabilized cells were then incubated in the dark for 30 min at room temperature with 20 μL PE-labeled anti-cytokine mAbs (IFN-γ, clone B27; TNF-α, clone MAB11; IL-4, clone MP4-25D2; IL-5, clone TRFK5; and IL-10, JES3-9D7 [e-Bioscience, San Diego, CA, USA]) in PBS permeabilization reagent.
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