Peripheral blood was collected from 13 KC patients (10 males, 3 females) and 13 volunteers (10 males, 3 females) by venous puncture and collected into preservative-free heparin. The PBMCs were isolated by means of Ficoll-Plaque Plus density gradient centrifugation (Amersham Biosciences, NJ, USA). For intracellular staining, PMBCs at a concentration of 1–10 million cells/mL were stained for 5 h with phorbol 12-myristate 13-acetate (PMA)/ionomycin at 50 ng/mL and 1 μg/mL, respectively. 4 μL of BD GolgiStop™ was added for every 6 mL of cell culture and mixed thoroughly. The PBMCs were then fixed in BD Cytofix™ Fixation Buffer and made permeable with BD Phosflow™ Perm/Wash Buffer. The percentages of TH1 and TH2 in CD4+ T cells were obtained through flow-cytometric analysis using the BD Human TH1/TH2/TH17 Phenotyping Kit, which has a four-color cocktail of fluorescent antibodies specific for Human CD4 (PERCP-CY5.5 Clone: SK3), IFN-γ (for TH1, FITC Clone: B27), IL-4 (for TH2, APC Clone: MP4-25D2), and IL-17A (for TH17, PE Clone: N49-653).
Ficoll plaque plus density gradient
Manufactured by Cytiva
Sourced in United States, United Kingdom
Ficoll-Plaque Plus is a sterile, pyrogen-free density gradient medium used for the isolation and purification of mononuclear cells from whole blood or other cell suspensions.
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Lab products found in correlation
2 protocols using ficoll plaque plus density gradient
1
Characterizing T-cell Subsets in Keratoconus
2
PBMC Cytokine Response to PM2.5 Exposure
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Blood was collected from 8 of the above-mentioned volunteers, and PBMCs were isolated from each sample using Ficoll-Plaque Plus density gradient centrifugation (Amersham Biosciences, Amersham, UK). The cells in each sample were adjusted to a concentration of 2 × 106 cells/mL, and 0.5 mL of cell suspension from each sample was incubated with 0.1 μg/mL PM2.5, 10 μg/mL PM2.5 or phosphate-buffered saline for 24 hours at room temperature. At the end of incubation, the samples were centrifuged at 1,500 g for 15 minutes at 4ºC, and the levels of IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-17a, IL-33, eotaxin, TGF-β1, TGF-β2, TGF-β3 and IFN-γ in the supernatants, were assessed using Luminex bead array technology as mentioned above.
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