Anti-CD3 (BD Biosciences; cat. no. 561798) and anti-CD28 (BD Biosciences; cat. no. 562764) antibodies were coated in each well on day 1, followed by addition of anti-IFN (BD Biosciences; cat. no. 551506) and anti-IL-4 (BD Biosciences; cat. no. 555090). Purified CD4+ T cells were seeded into a 24-well plate at a density of 1.5×106 cell per well in a 1 ml volume. Subsequently, 10 ng/ml IL-7 (PeproTech, Inc.) and 100 µM STAT5 inhibitor (Merck KGaA) were added to the culture, and incubated for 72 h. The following culture groups were obtained: Negative control (NC) group, Anti-CD3 + anti-CD28 + DMSO; NC + IL-7 group, Anti-CD3 + anti-CD28 + IL-7 + DMSO; NA group, Anti-CD3 + anti-CD28 + DMSO; NA + IL-7 group, Anti-CD3 + anti-CD28 + IL-7 + DMSO; NA + IL-7 + STAT5 inhibitor group:, Anti-CD3 + anti-CD28 + IL-7 + STAT5 inhibitor. The cells in each group were detected by a flow cytometer (FACS Calibur; BD Biosciences) and analyzed using FlowJo 7.6.5 software (FlowJo LLC).
Interleukin 7 (il 7)
Manufactured by Merck Group
Sourced in United States
IL-7 is a recombinant human interleukin-7 protein used in laboratory research. It is a cytokine that plays a key role in the development and maintenance of T cells. IL-7 supports the survival and proliferation of T cell progenitors and mature T cells.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Merck Group (3 mentions)
Facscalibur, by BD (2 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (2 mentions)
Il 15, by Merck Group (2 mentions)
Anti b220 microbeads, by Miltenyi Biotec (2 mentions)
Anti cd3, by BD (1 mentions)
Anti cd28, by BD (1 mentions)
Anti cd3, by Merck Group (1 mentions)
Anti cd28, by Merck Group (1 mentions)
Stat5 inhibitor, by Merck Group (1 mentions)
Anti il 4, by BD (1 mentions)
Interleukin 2 (il 2), by Roche (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Insulin transferrin sodium selenite supplement, by Merck Group (1 mentions)
X vivo 15 medium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Il 33, by Merck Group (1 mentions)
10 protocols using interleukin 7 (il 7)
1
CD4+ T Cell Activation and STAT5 Inhibition
2
Quantifying Latent HIV Infection in CD4+ T-cells
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Latent infection in the sorted, non-proliferating CD4+ T-cells (eFluor670hiEGFP−) was determined by comparison of stimulated with un-stimulated T-cells sorted from APC-T-cell co-cultures (control). 1x105 sorted CD4+ T-cells were stimulated with immobilized anti-CD3 (7 μg/ml; Beckman Coulter), in RF10 media supplemented with CD28 (5 μg/mL; BD Biosciences), IL-7 (50 ng/mL; Sigma, St Louis, MO, USA), IL-2 (5U/mL; Roche), with (post-integrated latency) or without (total latency: pre- and post-integrated latency) integrase inhibitor L8 (1 μM; Merck, White House Station, NJ, USA). The concentration of L8 was determined previously by titration of L8 in phytohaemagglutinin (PHA; 10 μg/mL) activated PBMC infected with R5-EGFP virus at an MOI of 0.5, same concentration usedin co-cultures, and showed productive infection was completely blocked at 1 μM. This concentration used for all subsequent experiments. Cells were harvested after 72 h of stimulation and EGFP expression was quantified on the FacsCalibur (BD BioSciences).
In some experiments PHA (10ug/mL) and IL-2 (10 U/mL) stimulated feeder PBMC were used to activate T-cells as a measure of inducing virus replication form latency, as described previously [14 (link)].
In some experiments PHA (10ug/mL) and IL-2 (10 U/mL) stimulated feeder PBMC were used to activate T-cells as a measure of inducing virus replication form latency, as described previously [14 (link)].
3
Isolation and Culture of T-ALL Blasts
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Bone marrow or peripheral blood samples from untreated children initially diagnosed with T-ALL were collected from the Czech Pediatric Hematology Centers. To be able to perform the here described experiments, only patients with the blast percentage higher than 80% and with a high cellularity were included. Within 24 h after aspiration, the mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, UK). All the samples were obtained with the informed consent of the children’s parents or guardians as well as the approval of the Ethical Committee of the University Hospital Motol, Prague, Czech Republic, study no. 201528848A. All experiments were performed in accordance with relevant guidelines and regulations. The isolated blasts were frozen in 90% fetal calf serum and 10% DMSO. After thawing, the blasts were maintained in X-VIVO™ 15 Medium with l -glutamine and gentamicin supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), IL-7 (50 ng/mL) and insulin-transferrin-sodium selenite supplement (Sigma-Aldrich, St Louis, MO, USA).
4
Isolation and Culture of Murine ILC2s
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Enriched kidney lymphocyte fractions were stained with anti-lineage cocktail (CD3ε, Ly-6G/Ly-6C (Gr-1), CD11b, CD45R (B220), Ter-119), anti-CD127 (IL-7Rα), anti-CD25 (IL-2Rα), anti-CD4, and anti-ST2 antibodies (Biolegend, San Diego, CA). Cells were sorted by SH800S cell sorter (SONY, Tokyo, Japan) with excluding dead cells by using 7-AAD. Gating strategy to sort ILC2s was lineage- CD127 + CD4- ST2 + CD25 + fraction (Supplementary Fig. 1 ), and the sorted ILC2 purity was routinely > 95%. These cells were cultured at 37 °C 5% CO2 in RPMI-1640 containing with 10% heat-inactivated FBS (Sigma, St. Louis, MO), penicillin/streptomycin, 50 μM 2-ME, 20 mM HEPES–KOH (pH7.53), 1 mM sodium pyruvate, 1 × non-essential amino acids (Wako). These cells were cultured with recombinant murine IL-2, IL-7, and IL-33 (each 10 ng/ml, Biolegend). To collect ILC2 supernatant, sorted ILC2s (1 × 105 cells/ml) were cultured for 3 days with IL-2, IL-7 and IL-33 in the presence or absence of DMSO, GSK360A (50 μM, Sigma) or FG-4592 (50 μM, Cayman chemicals, Ann Arbor, MI), and then collected supernatant filtered through 0.22-μm PVDF membrane.
5
Measuring STAT5 Phosphorylation in CD8+ T-cells
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Isolated blood-derived CD8+ T-cells or IH-lymphocytes (0.5x106/ml) were incubated with IL-7 (0.01–10 ng/ml) and/or IL-2 (100 ng/ml) and IL-15 (10 ng/ml, Sigma Aldrich, St. Louis, MO, USA) for 15 minutes at 37°C and phosphorylation of STAT5 (pSTAT5) was measured by flow cytometry, as described previously using the anti-pSTAT5 pY694 alexafluor 488 antibody (5μl/100μl cells, clone 47/STAT5(pY694), AB_399881 , BD Phosflow, BD Bioscience) with fixation in 4% paraformaldehyde and permeabilization in 100% cold methanol [32 (link)] (average age of controls was 39 ± 13, HCV+ individuals was 43 ± 12). The expression level of pSTAT5 in CD8+ T-cell subsets was determined after cells were stained for phenotypic markers using CD45RA-APC and CCR7 antibodies (average age of controls was 35 ± 11, HCV+ individuals was 56 ± 10). To distinguish CD8+ T-cells from other IH-lymphocytes, 5μl CD8-PeCy5 was added with the pSTAT5 antibody. The autofluorescence of IH-lymphocytes is higher than blood-derived cells, and this was taken into account during data analysis [38 (link)].
6
Xenograft Model of Lung Cancer Resistance
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Female nude BALB/c mice, aged 4–5 weeks, weighing 16–18 g, were procured from Southern Medical University and lodged in an SPF room under controlled conditions, including a 12 h light-dark cycle, a temperature of 23 ± 3°C, and a relative humidity of 55 ± 15%. The Institutional Animal Care and Use Committee of Zhujiang Hospital of the Southern Medical University approved by the animal protocol.
The xenograft models of human lung cancer DDP-resistant cell line A549/DDP was established by subcutaneously injecting the cells (4 × 106) into the mice. When tumors were palpable, the mice were randomly divided into 4 groups (n = 8) and treated with 0.1 mL/10 g twice a week intraperitoneally with a control vehicle (PBS), IL-7 (2 μg/injection; eBioScience, San Diego, CA, USA), DDP (5 mg/kg; Sigma, St. Louis, MO, USA), and IL-7 and DDP as described hereinbefore. The tumor sizes were measured once in three days apart and the tumor volumes examined weekly once and were calculated as V (cm3) = width2 (cm2) × length (cm)/2.
The xenograft models of human lung cancer DDP-resistant cell line A549/DDP was established by subcutaneously injecting the cells (4 × 106) into the mice. When tumors were palpable, the mice were randomly divided into 4 groups (n = 8) and treated with 0.1 mL/10 g twice a week intraperitoneally with a control vehicle (PBS), IL-7 (2 μg/injection; eBioScience, San Diego, CA, USA), DDP (5 mg/kg; Sigma, St. Louis, MO, USA), and IL-7 and DDP as described hereinbefore. The tumor sizes were measured once in three days apart and the tumor volumes examined weekly once and were calculated as V (cm3) = width2 (cm2) × length (cm)/2.
7
Isolation and Culture of Murine B Cells
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Bone marrow B cells were isolated from tibias, femurs, and humeri of ROSAerISCEIMycI/IIghI/I and ROSAerISCEIMycI/IIghI/IAID−/− mice at 6–8.5 mo of age by immunomagnetic enrichment with anti-B220 MicroBeads (Miltenyi Biotec). Cells were pooled and cultured at 2.0 × 106 cells/ml in the presence of IL-7 (5 or 10 ng/ml; Sigma-Aldrich) and tamoxifen (1 µM; Sigma-Aldrich) in complete RPMI. On day 1, cells were collected for IL-7 washout and replated in fresh complete RPMI with 1 µM tamoxifen. On day 2, cultures were harvested, and cell pellets were snap-frozen on dry ice.
8
Retroviral Transduction of Pro-B Cells
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Pro–B cells were isolated from tibias, femurs, and humeri of RAG2−/−MycI/I mice at 4–10 wk of age by immunomagnetic enrichment with anti-B220 MicroBeads (Miltenyi Biotec). Cells were cultured at 2.0 × 106 cells/ml in the presence of IL-7 (5 ng/ml; Sigma-Aldrich) in complete RPMI (RPMI-1640 supplemented with l -glutamine [Gibco], sodium pyruvate [Gibco], antibiotic/antimycotic [Gibco], Hepes [Gibco], 55 µM β-mercaptoethanol [Gibco], and 10% fetal calf serum [HyClone]). IL-7 was replenished on day 2. On days 3 and 4, cell supernatants were replaced with retroviral supernatants resulting from cotransfection (Fugene-6; Roche) of BOSC23 cells with pCL-Eco and pMX-I-SceI-P2A-RAG2core-EGFP or pMX-I-SceI-EGFP plasmids 3 d before (Robbiani et al., 2008 (link)). Spinoculation was at 1,111 g for 1.5 h in the presence of 2.5 µg/ml polybrene, 5 ng/ml IL-7, and 20 mM Hepes. After 6–8 h at 37°C, on day 3, retroviral supernatants were replaced with original supernatants, whereas on day 4, cells were collected for IL-7 washout and replating in fresh complete RPMI. Cells were harvested after 2.5 d of IL-7 depletion, sorted for EGFP expression with a FACSAria instrument (BD), pelleted, and snap-frozen on dry ice. Samples infected with pMX-I-SceI-P2A-RAG2core-EGFP are referred to as RAG2core, and those infected with pMX-I-SceI-EGFP are referred to as RAG2−/−.
9
Expansion of Naive CD8+ T-Cells
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Naive CD8+ T-cells were isolated isolated from blood using RosetteSep human CD8+ T cell enrichment cocktail (Stemcell) and Ficoll (GE Healthcare Life Sciences) gradient centrifugation. Naive CD8+ T-cells were stimulated by anti-CD3+CD28 Dynabeads (ThermoFisher) in Biotarget medium (Biological Industry) supplemented with 4% human platelet lysate (Ultra-GRO-Advanced, AventaCell) with 100U/mL IL-2 (R&D System) for 48hrs. Activated CD8+ T-cells were maintained in medium containing 100 U/mL IL-2, 10 ng/mL IL15 and 10 ng/mL IL7 (R&D System). T-cells were restimulated every 14-20 days by feeder cells, which were freshly isolated peripheral blood mononuclear cells (PBMC) and irradiated at 30 Gy. PBMC and T-cells were resuspended at a ratio of 2:1 in the medium with concentrations of 100U/mL IL-2, 10 ng/mL IL7, 10 ng/mL IL15 and 1.5 μg/mL of Lectin from Phaseolus vulgaris (Sigma-Aldrich). Blood collection protocol was approved by NUS IRB. Informed consent was obtained from all donors.
10
Optimized Neuronal Differentiation Protocol
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