For surface staining, cells were resuspended in HBSS+BSA+NaN3 (Quality Biological) with properly diluted antibodies. For intracellular staining, cells were fixed with 4% PFA in PBS (Ted Pella) for 30 min and then permeabilized with 0.5% Triton X-100 (Sigma) in PBS with 0.1% IgG-free BSA (Jackson Immunoresearch). IL-2 secretion was detected with a cytokine secretion assay (Miltenyi). Live cells were gated based on exclusion of 7AAD (eBioscience) or Live Dead Fixable dyes (Invitrogen). Data collected on a FACS Calibur or FACS Canto II (BD) were analyzed in FlowJo (Treestar). For NFATc1 localization, data were collected with an ImagestreamX Mark II imaging flow cytometer (Amnis/EMD Millipore). Data were analyzed with IDEAS (Amnis/EMD Millipore). Images were taken at 603 magnification.
Phosphate buffered saline (pbs)
Manufactured by Ted Pella
Sourced in United States
PBS (Phosphate-Buffered Saline) is a common buffer solution used in various laboratory applications. It is a balanced salt solution composed of sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate. PBS maintains the pH and osmolarity of the solution, providing a suitable environment for biological samples and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Cytokine secretion assay, by Miltenyi Biotec (1 mentions)
Igg free bsa, by Jackson ImmunoResearch (1 mentions)
Live dead fixable dye, by Thermo Fisher Scientific (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Ultramicrotome, by Leica (1 mentions)
Em208 transmission electron microscope, by Philips (1 mentions)
2 protocols using phosphate buffered saline (pbs)
1
Immunophenotyping and Cytokine Secretion
2
Brain Ultrastructural Analysis of Mice
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The mice were sacrificed, as mentioned above. The brains were removed, and the SN was dissected and processed with 3% glutaraldehyde (Tianjin Hengxing Chemical Reagent Co., Ltd., Tianjin, China), 1.5% paraformaldehyde (Tianjin Hengxing Chemical Reagent Co., Ltd., Tianjin, China), and 0.1 M PBS (Sigma- Gibco, MO, United States, Cat# 70011069) at 4°C per 24 h each. This procedure was followed by post-fixations in 1% osmium tetroxide and 1.5% potassium ferrocyanide at 4°C for 1.5 h. After washing with PBS, the samples were dehydrated in a graded series of ethanol (75%, 95%, 100% v/v) and embedded in an Epon-Araldite solution (Ted Pella, Redding, CA, United States) at 60°C for 72 h. Then, the sections were cut at 100 nm thickness using an ultramicrotome (Leica, Wetzlar, Germany) and imaged under an EM208 transmission electron microscope (Philips, Eindhoven, the Netherlands).
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