Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples collected in tubes with EDTA by conventional ficoll-uropoline density gradient centrifugation. PBMCs were then washed and resuspended in RPMI1640 medium supplemented with 5% FBS, penicillin (100 U/ml) – streptomycin (100 μg/ml) and 2-mercaptoethanol (5 × 10− 5 M) (all purchased from SigmaAldrich, Saint Louis, MO, USA). Cells (5 × 105 / 0.5 ml) were cultured for 48 h in the absence (control) or presence of IL-2 (100 U/ml) (BD Biosciences, San Jose, CA, USA), LPS (1 μg/ml) or PMA (50 ng/ml) and ionomycin (500 ng/ml, all purchased from Sigma-Aldrich). PBMCs treated in this way were analyzed for the expression of SIRT1, SOD2 and HSP70 (surface and intracellular). The intracellular expression of TNF and IFN-γ, was studied in PBMCs (5 × 105 / 0.5 ml) cultured in the absence (control) or presence of IL-2 (100 U/ml) (BD Biosciences, San Jose, CA, USA), LPS (1 μg/ml) Sigma-Aldrich, Saint Louis, MO, USA) or PMA (50 ng/ml) and ionomycin (500 ng/ml) (SigmaAldrich, Saint Louis, MO, USA) for 5 h. Simultaneously, Golgi Stop reagent (0.5 μl / well in 0.5 ml of medium, BD Biosciences, San Jose, CA, USA) was added to PBMC cultures (5 × 105 / 0.5 ml) to stop extracellular export of cytokines. Then PBMCs were collected and washed with 1 ml of BD Staining Buffer.
Interleukin 2 (il 2)
Manufactured by BD
Sourced in United States, United Kingdom, Germany
IL-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells and natural killer cells. It is a key component in the immune response, contributing to the growth and differentiation of these cell types.
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Lab products found in correlation
Ifn γ, by BD (35 mentions)
Tnf α, by BD (21 mentions)
Interleukin 4 (il 4), by BD (17 mentions)
Il 10, by BD (17 mentions)
Interleukin 6 (il 6), by BD (15 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (10 mentions)
Il 12, by BD (8 mentions)
Anti cd28, by BD (7 mentions)
Il 1β, by BD (7 mentions)
Ionomycin, by Merck Group (6 mentions)
Il 17, by BD (6 mentions)
Facscalibur, by BD (5 mentions)
Il 15, by Thermo Fisher Scientific (5 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (5 mentions)
Anti cd3, by BD (5 mentions)
Penicillin, by Merck Group (4 mentions)
Tnf α, by R&D Systems (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Tgf β, by BD (4 mentions)
Brefeldin a, by Merck Group (4 mentions)
140 protocols using interleukin 2 (il 2)
1
Isolation and Activation of PBMCs
2
PBMC Activation and Cytokine Expression
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Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples collected in tubes with EDTA by conventional ficoll-uropoline density gradient centrifugation. PBMCs were then washed and resuspended in RPMI1640 medium supplemented with 5% FBS, penicillin (100 U/ml) – streptomycin (100 μg/ml) and 2-mercaptoethanol (5 × 10− 5 M) (all purchased from Sigma - Aldrich, Saint Louis, MO, USA). Cells (5 × 105 / 0.5 ml) were cultured for 48 h in the absence (control) or presence of IL-2 (100 U/ml) (BD Biosciences, San Jose, CA, USA), LPS (1 μg/ml) or PMA (50 ng/ml) and ionomycin (500 ng/ml, all purchased from Sigma-Aldrich). PBMCs treated in this way were studied for the expression of SIRT1, SOD2 and HSP70 (surface and intracellular). The intracellular expression of TNF and IFN-γ, was studied in PBMCs (5 × 105 / 0.5 ml) cultured in the absence (control) or presence of IL-2 (100 U/ml) (BD Biosciences, San Jose, CA, USA), LPS (1 μg/ml) (Sigma-Aldrich, Saint Louis, MO, USA) or PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma - Aldrich, Saint Louis, MO, USA) for 5 h. Simultaneously, Golgi Stop reagent (0.5 μl / well in 0.5 ml of medium, BD Biosciences, San Jose, CA, USA) was added to PBMC cultures (5 × 105 / 0.5 ml) to stop extracellular export of cytokines. Then PBMCs were collected and washed with 1 ml of BD Staining Buffer.
3
Sézary Syndrome Patient Cell Study
4
Isolation and Activation of CD4+ T Cells for HIV Co-cultivation
5
Isolation and Activation of CD4+ T Cells for HIV Co-cultivation
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PBMC were isolated from heparinized blood using a Ficoll-Paque Plus density gradient (Sigma). CD4+ T lymphocytes were then isolated by positive selection using anti-CD4 microbeads (Miltenyi Biotec) (Tugizov et al., 2012 (link)). PBMC were activated with 2.5 μg/ml phytohemagglutinin (Sigma) and 1 μg/ml interleukin-2 (BD Biosciences) for 3 days. For cocultivation of activated PBMC with polarized epithelial cells containing HIV, ~ 106 lymphocytes were washed twice and added to AP or BL surfaces of epithelial cells as described in our recent work. The ratio of lymphocytes to epithelial cells was ~ 2:1. After 4 h, medium and lymphocytes were collected by pipeting and centrifuged for 10 min at 1200 rpm. PBMC were grown for 3–12 days and analyzed for HIV-1 infection by ELISA p24.
6
Cytokine Quantification in Intestinal Samples
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Serum samples and supernatants of the small intestine were used for the quantification of cytokines. Specifically, ELISA kits (R & D Systems, San Jose, CA, USA) were used to determine IFNγ, IL-6, IL-17, and IL-23 following the manufacturer’s instructions. In addition, IL-2 (detectable minimal dose [DMD] 0.1 pg/mL), IL-4 (DMD 0.03 pg/mL), IL-10 (DMD 6.8 pg/mL), and TNFα (DMD 0.9 pg/mL) (Becton Dickinson and Company, San Jose, CA, USA) were evaluated by flow cytometry (BD FACSCantoTM II, San Jose, CA, USA) and date were analyzed used the FCAP array Software (BD San Jose, CA, USA). These assays provided a method of capturing soluble analytes or a set of analytes with beads of known size and fluorescence.
7
Modulation of T Cell Responses by DC Stimuli
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Naïve CD4+ T cells were stimulated with anti-CD3/anti-CD28 activation beads (1:1 ratio) (ThermoFisher) and 20 IU of IL-2 (Becton Dickinson) for 7 days in the presence of supernatants derived from mDC prestimulated with either 50 µg/ml of curdlan or glucan-mp. CD4+ T cell culture supernatants were subsequently harvested to evaluate IL-17 and IFNγ secretion.
8
Multiparametric Flow Cytometry Panel
9
SARS-CoV-2-Specific T Cell Immune Response Profiling
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Cryopreserved cells were thawed, rested for at least 2 h in AIM-V medium and then cultured in the presence of 2 μg/ml PepTivator® SARS-CoV-2 Prot_S Complete for 16–20hrs in 96-wells U-bottom plates using 2 × 106 PBMCs per well. As a positive control, Cell Stimulation Cocktail (Thermo Fisher) was used and added for the last 4hrs. Surface and intracellular staining of PBMCs was performed according to routine protocols and using appropriate combinations of antibodies for the detection of CD3, CD4, CD8, CD45RA, CD107a, CD137, CD154, CCR7, CXCR5, PD-1, IFNγ, TNFα, IL-2, Granzyme B (all Becton Dickinson, San Diego, USA), and Fixable Viability Dye (Thermo Fisher, Waltham, USA). For intracellular staining of cytokines, Golgi transport was inhibited by Protein Transport Inhibitor Cocktail (Thermo Fisher) for 4 h prior to intracellular staining. For both approaches, surface staining was performed for 20 min at 4 °C. Afterwards, samples were fixed and permeabilized using the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Biosciences). Intracellular staining was performed in Perm/Wash buffer for 30 min at 4 °C. Samples were acquired on a FACSLyric instrument (BD Biosciences) and analyzed with FlowJo software version 10.5.3 (FlowJo LLC).
10
Multiparametric Flow Cytometry Analysis of PBMCs
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PBMCs were then surface-stained with the following monoclonal antibodies to: CD3 (PerCP, Bio Legend, San Diego, USA), CD4 (Alexa Flour 700, Becton Dickinson, San Diego, CA, USA), CD8 (APC-H7, Becton Dickinson, San Diego, CA, USA), CD56 (FITC Becton Dickinson, San Diego, CA, USA). Next, monoclonal antibodies were washed out and stained for live/dead discrimination with an aminereactive dye (Alexa Fluor 350 carboxylic acid, succinimidyl ester, Invitrogen, Carlsbad, CA, USA). After 10 min of incubation at room temperature, cells were fixed and permeabilized using a fix and perm kit® (An Der Grub Bio Research GmbH, Vienna, Austria). Afterwards, intracellular staining was conducted with antibodies against interferon-γ (IFN-γ, BV605 Becton Dickinson, San Diego, CA, USA), interleukin-2 (IL-2, BV510 Becton Dickinson, San Diego, CA, USA) and tumor necrosis factor-α (TNF-α, BV421 Becton Dickinson, San Diego, CA, USA). Measurements were carried out on a flow cytometer LSR II (Becton Dickinson, San Diego, CA, USA) and FACSDIVA acquisition/analysis software (Becton Dickinson, San Diego, CA, USA) was used for data analysis.
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