Sixteen HIV+ viremic donors were used throughout in this study. Blood mononuclear cells were maintained in complete RPMI (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biowest), 100 U/ml penicillin (BioConcept), and 100 µg/ml streptomycin (BioConcept). 293T cells were maintained in DMEM (Invitrogen) supplemented with 10% (v/v) FBS, 100 U/ml penicillin (BioConcept), and 100 μg/ml streptomycin (BioConcept). Cells were kept at 37°C, 5% CO2 for the following experiments.
Streptomycin
Manufactured by BioConcept
Sourced in Switzerland
Streptomycin is a broad-spectrum antibiotic used to treat various bacterial infections. It is a product of the bacterium Streptomyces griseus and functions by inhibiting protein synthesis in susceptible microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by BioConcept (31 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (12 mentions)
Golgiplug, by BD (6 mentions)
Cytofix cytoperm, by BD (5 mentions)
Fetal calf serum (fcs), by BioConcept (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Aqua live dead stain kit, by Thermo Fisher Scientific (3 mentions)
L glutamine, by BioConcept (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Cytof2, by Standard BioTools (2 mentions)
Fungizone, by BioConcept (2 mentions)
Complete rpmi medium, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Violet live dead stain kit, by Thermo Fisher Scientific (1 mentions)
Anti cd3 anti cd28 beads, by Thermo Fisher Scientific (1 mentions)
34 protocols using streptomycin
1
HIV+ Donors Cell Culture
2
T Cell Activation and Culture Conditions
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The culture medium consisted of RPMI 1640 with Hepes (Gibco) supplemented with 5% heat-inactivated human serum (Swiss Red Cross, Basel, Switzerland), 1% Glutamax (Gibco), penicillin (50 U/ml) and streptomycin (50 μg/ml) (BioConcept), and IL-2 (50/250 IU/ml) (Roche). The glucose medium consisted of glucose-free RPMI 1640 (Gibco) supplemented with 5% heat-inactivated dialyzed FBS (Gibco), 1% Glutamax (Gibco), penicillin (50 U/ml) and streptomycin (50 μg/ml) (BioConcept), IL-2 (50 IU/ml) (Roche) and glucose (Sigma-Aldrich). The fatty acid-free medium consisted of PBS (pH 7.4) supplemented with 0.5% fatty acid-free BSA (Sigma-Aldrich) and 1 mM EDTA. T cells were cultured in a 96-well plate with a density of 0.25 × 105 to 1 × 105 cells per well in a total volume of 200 μl of cell culture medium. T cells were polyclonally activated using ImmunoCult Human CD3/CD2/CD28 T Cell Activator (1:100) (Stemcell Technologies).
3
Culturing Reporter Jurkat Cell Line
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Cells were cultured in RPMI (Gibco; Life Technologies) containing 10% heat-inactivated FBS (Institut de Biotechnologies Jacques Boy), 100 IU/ml penicillin and 100 μg/ml streptomycin (Bio Concept). PD-1/NFAT reporter Jurkat recombinant cell line (BPS Bioscience #60535) were cultured in DMEM (Life Technologies; #41965–039) containing 10% heat-inactivated FBS (Institut de Biotechnologies Jacques Boy), 100 IU/ml penicillin and 100 μg/ml streptomycin (Bio Concept), 1mg/ml Geneticin (Thermofisher; #10131027), and 200 μg/ml of Hygromycin B (Chemie brunschwig; #MED30-240-CR).
4
Cytokine Profiling of Stimulated PBMCs
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PBMCs were stimulated overnight in complete media (RPMI (Invitrogen), 10% fetal calf serum (FCS; Invitrogen), 100 µg/ml penicillin, 100 unit/ml streptomycin (BioConcept)) with ESAT-6 and CFP-10 peptide pools (1 µg/ml) or with Staphyloccocus enterotoxin B (SEB; 250 ng/mL) or unstimulated in the presence of Golgiplug (1 μl/ml; BD) as previously described [95 (link)]. At the end of the stimulation period, cells were washed and stained (20 min; 4°C) for dead cells using the Aqua LIVE/DEAD stain kit (Invitrogen), washed and stained (20 min; 4°C) with mAbs to CD3, CD4, CD8 and CD45RA. Cells were then permeabilized (30 min; 20°C) (Cytofix/Cytoperm, BD) and stained (20 min; 20°C) with mAbs to TNF-α, IFN-γ, IL-2, IL-4, IL-5 and IL-13.
5
Cytokine Profiling of Activated T Cells
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Freshly isolated blood mononuclear cells (106 cells) were stimulated overnight as previously described (Perreau et al., 2012 (link)) in complete RPMI medium (10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin [Bioconcept]). Blood mononuclear cells were stimulated with 1 µg/ml Ad5 vector or CMV lysat (Advanced Biotechnologies INC) or 5 × 107 CFU/ml of individual bacteria or bacteria pool depending on the experiment. As a positive control, cells were stimulated with anti-CD3/anti-CD28 beads (Invitrogen). Cells were incubated for 18 h in 1 ml of complete media containing 1 µl/ml brefeldin A (Golgiplug; BD). At the end of the stimulation period, dead cells were stained (4°C, 15 min) using the violet LIVE/DEAD stain kit (Invitrogen). Cells were washed, permeabilized (Cytofix/Cytoperm solution; BD), and stained (4°C, 15 min) with anti–CD3-APC-H7, anti–CD4-ECD, anti–CD8-PerCP, anti–IFN-γ-AF700, anti–IL-17A-AF647, anti–TNF-PE-CY7, and anti–IL-2-PE as previously described (Perreau et al., 2012 (link)). Frequencies of cytokine-producing CD4 T cells were assessed by flow cytometry.
6
Type I IFN Induction by F. novicida
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One day before infection, ISRE-L929 reporter cells (a gift from D.M. Monack, Stanford University) were seeded into black 96-well plates with micro-clear bottom (Greiner) at a density of 1 × 105 cells per well in DMEM (Sigma) with 10% v/v FCS and penicillin (100 IU ml−1)/streptomycin (100 μg ml−1) (both BioConcept). BMDMs were seeded into 96-well plates at a density of 5 × 104 cells per well and infected with F. novicida at a multiplicity of infection of 100 as described above. After 10 h of infection, type I IFN production was measured with the Bright-Glo Luciferase Assay System (Promega) as previously described80 (link).
7
Culturing HeLa Cells with Chloroquine and Isolating Murine Hepatocytes
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HeLa cells (European Cell Culture Collection) were cultured in minimum essential medium with Earle’s salts (MEM EBS; BioConcept, 1‐31F01‐I), supplemented with 10% FCS (GE Healthcare), 100 U penicillin (BioConcept), 100 μg/ml streptomycin (BioConcept), and 2 mM L-glutamine (BioConcept). Cells were cultured at 37 °C and 5% CO2 and split using Accutase (Innovative Cell Technologies) diluted 1:2 in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Chloroquine treatment was performed in MEM complete supplemented with 10 μM Chloroquine (Sigma-Aldrich C6628).
Primary murine hepatocytes were isolated and cultured as described elsewhere3 (link). For infection of cells, salivary glands of infected Anopheles stephensi mosquitoes from 16–26 days after blood feeds were isolated and homogenized to release sporozoites. Sporozoites were incubated with cells in medium containing 25 μg/ml Amphotericin B (Amresco E437) for 2 h. Subsequently, cells were rinsed and incubated in MEM EBS complete containing 2.5 μg/ml Amphotericin B.
Primary murine hepatocytes were isolated and cultured as described elsewhere3 (link). For infection of cells, salivary glands of infected Anopheles stephensi mosquitoes from 16–26 days after blood feeds were isolated and homogenized to release sporozoites. Sporozoites were incubated with cells in medium containing 25 μg/ml Amphotericin B (Amresco E437) for 2 h. Subsequently, cells were rinsed and incubated in MEM EBS complete containing 2.5 μg/ml Amphotericin B.
8
RPMI-based Cell Culture Protocol
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Cells were cultured in RPMI (Gibco; Life Technologies) containing 10% heat-inactivated FBS (Institut de Biotechnologies Jacques Boy), 100 IU/ml penicillin and 100 μg/ml streptomycin (Bio Concept), hereafter referred to as complete RPMI (cRPMI).
9
Histone Deacetylase Inhibitor Protocols
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Vorinostat (Merck Research Laboratory, USA), romidepsin (Cellgene, USA), panobinostat (Novartis, Switzerland), givinostat (Italfarmaco; Italy), belinostat (Topotarget and Spectrum Pharmaceuticals, Denmark), and bryostatin were obtained from Hölzel Diagnostika (Germany) and resuspended in dimethyl sulfoxide (DMSO). Cells were cultured in RPMI (Gibco, Life Technologies) containing 10% heat-inactivated fetal bovine serum (FBS) (Institut de Biotechnologies Jacques Boy), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Bio Concept).
10
Culturing Tumor Cell Lines for Assays
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Tumor cell lines were established from tumor fragments and cultured in R10 medium (RPMI 1640 complemented with 10% FBS, 100 mM HEPES (Gibco), 100 IU ml−1 of peninicillin and 100 μg ml−1 of streptomycin (Bio-Concept)) at 37 °C at 5% CO2. Culture medium was replenished every 2–3 d, and cultures were split when confluent. To this end, tumor cells were gently detached with Accutase (Thermo Fisher Scientific) and split, and R10 medium was fully replenished. The day before any co-culture assay (screening assay described below), tumor cells were incubated for 24 h in R10 medium supplemented with 200 ng ml−1 of IFNγ (Miltenyi Biotec).
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