PBMCs or isolated CD8+ T cells were cultured and stimulated with soluble Purified NA/LE Mouse anti-human CD3 (UCHT1, 3 μg/mL) and anti-human CD28 (CD28.2, 1 μg/mL) or PMA/Ionomycin (Cell stimulation cocktail, BioLegend) in the presence of the protein transport inhibitor Brefeldin A (BD Biosciences) for 5 hours. After fixation and permeabilization (BD Cytofix/Cytoperm), intracellular cytokine staining was performed according to our previous methods.32 33 In some experiments, PBMCs were stimulated with anti-CD3/CD28 antibodies and treated with different cytokines such as IL-16 (R&D, 500 ng/mL) for 72 hours. Then, CD8+ T cells were subjected to flow cytometry and RT-PCR for the quantification of CD160. In long-term cultures, every 72 hours, fresh media and cytokine/stimulation cocktails were added. Also, isolated B-CLL cells (1×106) were cultured for 12 hours and then IL-16 levels were measured in the culture supernatant by the quantikine ELISA kit (R&D).
Il 16
Manufactured by R&D Systems
Sourced in United States
IL-16 is a cytokine that functions as a chemoattractant for CD4+ T cells. It is involved in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Cell stimulation cocktail, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Gasdermin e, by Abcam (1 mentions)
Caspase 3, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by BD (1 mentions)
Gasdermin d, by Abcam (1 mentions)
4 protocols using il 16
1
Stimulating and Analyzing CD8+ T Cells
2
Maternal Serum Biomarker Profiling
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Maternal venous blood samples were collected between 8 and 9 o'clock in the morning. The subjects were fasted prior to blood collection and they were at bed rest. The subjects did not exercise during the study.
To determine PlGF, IL 6, and IL16 in maternal serum, we used vacutainer tubes without additives and allowed samples to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 ×g. In order to determine serum sFlt-1 and sEng, we put the collected blood in refrigerator at 4°C for the night; then, the blood was centrifuged for 10 minutes at 1000 ×g. We stored the samples at −80°C until assayed. The laboratory personnel performing the assays were blinded to the clinical information of each subject. The concentrations of PlGF, sFlt1, sEng, IL-6, and IL-16 were determined by enzyme-linked immunosorbent assay (ELISA): PlGF was purchased from R&D Systems Inc. (DPG00), sFlt1 (MBS2601616) and sEng (MBS269385) reagents were purchased from My BioSource, IL-6 (EK0410) and IL-16 (EK0428) were purchased from Boster Biological Technology Co., Ltd., Pleasanton, USA, and each sample was run in duplicate. The blood pressure was taken in a semirecumbent position, with a supported arm and appropriately sized cuff using a manual sphygmomanometer.
To determine PlGF, IL 6, and IL16 in maternal serum, we used vacutainer tubes without additives and allowed samples to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 ×g. In order to determine serum sFlt-1 and sEng, we put the collected blood in refrigerator at 4°C for the night; then, the blood was centrifuged for 10 minutes at 1000 ×g. We stored the samples at −80°C until assayed. The laboratory personnel performing the assays were blinded to the clinical information of each subject. The concentrations of PlGF, sFlt1, sEng, IL-6, and IL-16 were determined by enzyme-linked immunosorbent assay (ELISA): PlGF was purchased from R&D Systems Inc. (DPG00), sFlt1 (MBS2601616) and sEng (MBS269385) reagents were purchased from My BioSource, IL-6 (EK0410) and IL-16 (EK0428) were purchased from Boster Biological Technology Co., Ltd., Pleasanton, USA, and each sample was run in duplicate. The blood pressure was taken in a semirecumbent position, with a supported arm and appropriately sized cuff using a manual sphygmomanometer.
3
Quantification of Cytokine Secretion
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Human cytokines secretion were quantified by ELISA kits, according to the manufacturer’s instructions, IL-1B (Thermo Fisher Scientific, (88-7261-77), IL-6 (BD, 555220), IL-16 (R&D, DY316), Caspase 3 (Invitrogen, BMS2012INST), Gasdermin E (AG-45B-0024-KI01), Gasdermin D (Abcam, ab272463), Cleaved Caspase-3caspase-3 (Abcam, ab220655). Before use, the samples were diluted 1/3 to 1/5 with their respective assay diluents. IL-18 was quantified using IL-18 Reporter HEK 293 Cells according to the manufacturer’s instructions (InvivoGen). HMGB1 was quantified by ELISA according to the manufacturer’s instructions (Novus Biologicals).
4
Transwell Assay for NK Cell Migration
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Transwell assay was conducted as previously indicated.15 In brief, NK cells were seeded in the upper layer of a Costar 24‐well Transwell plate (8 μm diameter). Next, 600 μL of RPMI 1640 supplemented with 10% FCS and 200 ng/mL IL‐16 (R&D Systems) was placed in the bottom layer of the Transwell plate. After incubation for 4 hours at 37°C, the cell number in each lower well was recorded twice, and the cell migration rate in regular medium was set as 100%. Migration due to chemotaxis was calculated as the percentage of the spontaneous migration.
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