DCs were stimulated with HIV‐1 Cap‐RNA58, R848, or a combination. As a positive control for T helper (TH) 1 skewing, DCs were stimulated with LPS (10 ng/ml; Sigma) and IFNγ (1000 U/ml; u‐CyTech) and with LPS and PGE2 (1 μM; Sigma) to induce TH2 skewing. After 48 h, DCs were collected, washed, and cocultured with isolated naïve CD4+ T cells from an allogeneic donor in a 1:4 ratio. DC‐T cell cocultures were performed in IMDM medium (Invitrogen) supplemented with 10% FCS, penicillin, and streptomycin (100 U/ml and 100 μg/ml, respectively; Thermo Fisher, IMDM complete) and in the presence of Staphylococcus aureus enterotoxin B (10 pg/ml; Sigma). On day 5, IL‐2 (10 U/ml; Chiron) was added and on day 11–13, resting T cells were restimulated using PMA (10 ng/ml; Sigma) and ionomycin (1 mg/ml; Sigma) for 6 h and brefeldin A (10 μg/ml; Sigma) for the final 4 h. Cells were fixed with 4% PFA, permeabilized with 0.1% saponin in PBS with 0.5% BSA, and stained with FITC‐conjugated anti‐IFNγ (1:5, 340449; BD Biosciences) and APC‐conjugated anti‐IL‐4 (1:25, 554486; BD Biosciences), indicative of TH1 and TH2 skewing, respectively. Flow cytometry was performed using FACSCanto II (BD Biosciences) and analyzed with FlowJo software v10.7.
Complete imdm
Manufactured by Merck Group
Sourced in Canada
Complete IMDM is a basal cell culture medium formulated for the growth and maintenance of various cell types. It provides the necessary nutrients and ingredients for supporting the metabolic requirements of cells in culture.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (2 mentions)
Facscanto 2, by BD (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Imdm medium, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Fitc conjugated anti ifn γ, by BD (1 mentions)
Staphylococcus aureus enterotoxin b, by Merck Group (1 mentions)
Mouse liver dissociation kit, by Miltenyi Biotec (1 mentions)
Dnase, by Merck Group (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Gentlemacs dissociator device, by Miltenyi Biotec (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Facscalibur, by BD (1 mentions)
Monensin, by Merck Group (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
7 protocols using complete imdm
1
Th1/Th2 Polarization of CD4+ T Cells by DC Stimulation
2
Isolation and Purification of Liver and Spleen Leukocytes
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The spleens and livers were flushed by heart perfusion with PBS. Single-cell suspension of splenocytes was prepared as described before (38 (link)). Liver leukocytes were either isolated using the mouse liver dissociation Kit and the gentleMACS dissociator device (both from Miltenyi Biotec) according to the manufacturer’s instructions or, alternatively, minced with scissors, suspended in 3 ml of digestion medium (IMDM-complete with 0.02 mg L−1 of Collagenase D and 0.01 mg L−1 of DNAse; both from Sigma-Aldrich), transferred to Falcon tubes, and incubated (30 min, 37°C) while the suspension was sheared frequently. After 30 min, 2 ml of fresh digestion medium was added, and the livers were incubated for an additional 15 min. Afterwards, 60 μl of 0.5 mol L−1 ethylenediaminetetraacetic acid (EDTA) was added, and samples were rested (5 min, 37°C) to stop enzymatic digestion. Cell suspension was passed through 100-μm cell strainers, washed with PBS, and afterwards passed through a 70-μm cell strainer, followed by centrifugation (300 rcf, 10 min) at room temperature (RT). Erythrocyte lysis was performed as described previously (38 (link)). The resulting pellet was resuspended in 10 ml of 35% EasyColl/PBS and centrifuged (1,800 rpm, 10 min, RT, no brake). Leukocyte pellet was resuspended in IMDM-complete and stored on ice.
3
Western Blot Analysis of LPA Signaling
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50.000 cells were seeded in each well of 12-well plates. The cells were grown for 24 hours in complete IMDM (Sigma-Aldrich), then in serum-free medium for 24 hours before stimulation with LPA with or without specific inhibitors as indicated in Results. After stimulation, cells were washed once with PBS, and scraped directly in Laemmli buffer (4% SDS, 20% Glycerol, 120 mM Tris–HCl, pH6.8, 0.006% bromphenol blue and 10% mercaptoethanol), and aliquots of 20 μg protein were separated on 10% polyacrylamide gels by electrophoresis under denaturing conditions. The proteins were transferred to nitrocellulose membranes using a semidry transfer system (Bio-Rad). The membranes were incubated with primary antibody in Tris-buffered saline containing 0.1% Tween 20 (TBST) with 5% non-fat dry milk or BSA overnight at 4°C. The blots were then washed three times in TBST and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. The blots were visualized with LumiGLO® (KPL, Gaithersburg, MD).
4
Detecting Cytokines in N. brasiliensis
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For detection of intracellular cytokines, MST or lung cells from N. brasiliensis infected mice were plated at 2×106 cells/well and stimulated at 37°C for 4 h with 50 ng/ml phorbol (myristate acetate), 250 ng/ml ionomycin and 200 µM monensin in complete IMDM (all purchased from Sigma-Aldrich). Cells were stained with extracellular markers [CD4 PercP Cy5.5 (RM4-5) and B220 FITC (RA3-6B2)], fixed for 30 min on ice in 2% (w/v) paraformaldehyde and permeabilised with 0.5% saponin buffer and stained with PE-labelled anti-mouse IL-4, IL-13, and IFN-γ for 30 min. Acquisition was performed using a FACSCalibur (BD Immunocytometry Systems, San Jose, CA, USA) and data were analysed using FlowJo software (Treestar, Ashland, OR, USA).
5
Quantification of Phosphorylated ERK
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50.000 cells were seeded in each well of 12-well plates. The cells were grown for 24 hours in complete IMDM (Sigma-Aldrich), then in serum-free medium for 24 hours before stimulation with LPA with or without specific inhibitors as indicated in Results. Cells were lysed with Bicine/CHAPS buffer with aqueous and DMSO inhibitor mixes (Protein Simple, Santa Clara, CA, USA) on ice. Lysates (0.1 mg/ml) were mixed with fluorescent pI Standard Ladder 3, Ampholyte premix G2, pH 5–8 separation gradient (Protein Simple) and loaded into capillaries in a Cell Biosensis Protein Simple NanoPro 1000 system according to the manufacturer’s instructions. Proteins were separated by capillary isoelectric focusing separation performed at 21 mW for 40 minutes and then immobilized with UV light exposure for 70 s. Anti-ERK1/2 antibody or anti-phospho ERK1/2 was then applied to the capillaries and probed with secondary anti-mouse IgG. The NanoPro machine performed automated wash steps with Wash buffer (Protein Simple). Primary antibody was incubated for 2 hours and secondary antibody for 1 hour. Signal was detected with Luminol and Peroxide (Protein Simple) and imaged with a CCD camera. Quantitation was performed with Compass software (Protein Simple). The method is described and validated by O’Neill et al. [32 (link)].
6
Culturing and Differentiating Innate Lymphoid Cells
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OP9-DL1 cells (provided by J.C. Zúñgia-Pflücker, Sunnybrook Research Institute, Toronto, Canada) were maintained in complete IMDM (Sigma-Aldrich). Rag1−/− NKp46+ ILC3s were sorted (CD45loCD90hi, KLRG1−, Nkp46+CCR6−) and cultured on OP9-DL1 stromal cells in complete IMDM in the presence of SCF (10 ng ml−1; eBioscience) and Flt3L (10 ng ml−1; eBioscience) for 24 h with additional cytokines as indicated. IL-7 (10 ng ml−1; eBioscience), IL-23 (50 ng ml−1; eBioscience), IL-12 (10 ng ml−1 for Fig. 6 D and 50 ng ml−1 for Fig. 6 C ; eBioscience), IL-15 (10 ng ml−1; BioLegend), and IL-18 (50 ng ml−1; BioLegend) were used as well. MNK cells were cultured as described (Allan et al., 2015 (link)). MNK-3 cells were sorted to select for NKp46+ cells and are referred to as NKp46+ MNK-3. NKp46+ MNK-3 were cultured in IL-7 (10 ng ml−1; eBioscience) and IL-15 (10 ng ml−1; BioLegend).
7
Lymph Node Proliferation Assay
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For radioactive proliferation assays, we activated 2 Â 10 5 lymph node cells per well with 60 μg/ml of OVA (Research Genetics) or MOG 35-55 (Genscript) with or without 5 μg/ml antibody to CD28 (37 N, Bioexpress) in complete IMDM (Sigma-Aldrich) in a 96-well plate in quadruplicates, cultured at 5% CO 2 .
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