Bone marrow was harvested from the femurs of 10-week-old C57BL/6J mice and single cell suspensions prepared as described previously [45 (link)]. The cells were then grown for 6 days in bone marrow differentiation media (DM) with medium exchange d4 of culture. DM comprises complete RPMI medium (Merck) containing 10% heat-inactivated fetal bovine serum (Biochrom GmbH), 1% Antibiotic-Antimycotic solution (Merck), 50 µM 2-mercaptoethanol (Merck) and was supplemented with L929 cell (ATCC CCL-1) conditioned media. L929 cell conditioned medium contains colony-stimulating factor 1 (CSF-1) and was produced by growing L929 cells in complete RPMI medium for 10 days. On d6 of culture, adherent cells were harvested and re-plated in 96F-well plates at 1 × 106 cells/mL and incubated overnight to allow cells time to adhere. The following day, macrophages were stimulated with 1, 10, 100, or 1000 µg/mL of either EcN or EcO lysate in a humidified incubator (37 °C, 5% CO2) for 24 h. Either lysate was prepared by passing the suspensions 3 times through a French Press, lyophilized, re-suspended in complete RPMI medium, and tested for sterility.
Complete rpmi medium
Manufactured by Merck Group
Sourced in Germany
Complete RPMI medium is a cell culture medium that provides a balanced formulation of nutrients, vitamins, and salts to support the growth and maintenance of a variety of cell types in vitro. It is a widely used medium in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Antibiotic antimycotic solution, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Heat inactivated fetal bovine serum, by Harvard Bioscience (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Il 1β, by Bio-Techne (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Neubauer chamber, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
11 protocols using complete rpmi medium
1
Macrophage Stimulation with Bacterial Lysates
2
Cytokine Profiling in Gut Tissue
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Next, Peyer’s patches and samples of colon tissue were cultured ex vivo, as described earlier [43 (link)]. Briefly, three distal Peyer’s patches and one three-millimeter punch biopsy from distal colons were collected, weighed and cultured in 500 µL of complete RPMI medium (Merck; Cat# R0883) containing 10% heat-inactivated fetal bovine serum (Biochrom GmbH, Berlin, Germany; Cat# S 0115) and 1% Antibiotic-Antimycotic solution (Merck) in a humidified incubator (37 °C, 5% CO2) for 48 h. The supernatants were collected and stored at −20 °C until analysis. Cytokines were measured in tissue culture supernatants using appropriate ELISA sets for murine TNF-α, IL-6, IL-1β, IL-33, and S100A8 (all Bio-Techne, Minneapolis, MN, USA; Cat# DY410, DY406, DY401, DY3626 and DY3059) according to the manufacturer’s instructions.
3
Culturing Breast Cancer Cell Lines
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Human breast cancer cell lines, triple-negative BT20 (ER−/PR−/HER2−) (ATCC-HTB19), triple-positive BT474 (ER+/PR+/HER2+) (ATCC-HTB20), and HER2-positive SKBR3 (ER−/PR−/HER2+) (ATCC-HTB30), were cultured in complete RPMI medium (Sigma-Aldrich), supplemented with 10% v/v fetal bovine serum (FBS), 2 mM l -glutamine, 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow at 37°C under 5% v/v CO2. Since these cell lines were adherent, cells were detached using 2× trypsin–EDTA (0.5%) (Fisher Scientific) for 10 min at 37°C, then centrifuged at 1,500 rpm for 5 min, and resuspended in complete RPMI. Cell number and viability were assessed by mixing an equal volume of the cell suspension and Trypan Blue (0.4% w/v) (Fisher Scientific) and by counting using a hemocytometer with a Neubauer chamber (Sigma-Aldrich).
4
Culturing Human Cancer and Immune Cell Lines
5
Macrophage Activation with LPS and CDNPs
6
Splenocyte Isolation for Cytokine Profiling
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To obtain splenocytes, spleens were extracted aseptically from immunized mice, pooled and homogenized in 10 mL of complete RPMI medium (Sigma) using a 5‐ml syringe plunger. The tissue was squeezed through a 70 μm cell strainer (BD FalconTM). The released cells were spun, and the pellet was resuspended in RPMI medium. To eliminate red blood cells, the pelleted cells were incubated with ACK lysing buffer (Gibco) for 3 min at 37 °C and washed two times with 25 mL complete medium. Triplicate cultures were seeded into 96 well U‐bottom plates at a density of 3 × 105 cells/well, in 200 μL medium and stimulated with 10 μg/mL of dengue antigen or PBS as a control. After incubation of cells for 48 h at 37 °C, 100 μL of supernatant was removed for Th1/Th2 and IL‐17 cytokine ELISA assays. The experimental procedure described by the manufacturer (Mouse Th1/Th2 and Th17 ELISA Ready‐SET‐Go kit; affymetrix eBioscience, USA) was then followed.
7
Cell Culture of Genetically Modified Lines
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Cell lines were obtained from the American Type Culture Collection. HEK-293T cells were cultured in complete Iscove’s modified Dulbecco’s medium (Sigma Aldrich) supplemented with 10% FCS and 2 mM Glutamax (Gibco) (cIMDM). RAJI and NALM6 were maintained in complete RPMI medium (Sigma Aldrich) supplemented with 10% FCS and 2 mM Glutamax (Gibco) (cRPMI). RAJI CD19 wild type (RAJI-19WT) cells were transduced with SFG vector for enhanced green fluorescence protein (eGFP) (RAJI-19GFP), and RAJI CD19 knockout (RAJI-19KO) cells were edited using CRISPR-Cas9. NALM6 FLUC cells were transduced with SFG vector to express firefly luciferin and a lipid anchored hemagglutinin (HA-GPI) tag.
8
CFBE41o− Cell Culture and Macrophage Differentiation
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CF epithelial cells, CFBE41o− which are homozygous for the ΔF508 mutation of the CFTR gene were routinely cultured in collagen/fibronectin‐coated flasks as previously described (Cullen et al., 2017 (link)). The U937 macrophage cells were maintained as a suspension culture in complete RPMI medium (Sigma‐Aldrich) supplemented with 1 mM sodium pyruvate, 10 mM 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) buffer (pH 7.0–7.6), 1 mg/100 units streptomycin/penicillin, 10% (v/v) fetal bovine serum (FBS) and 5 g/L d ‐glucose. U937 cells were plated in 24‐well plates and after 24 h were induced to differentiate by the addition of 15 ng/ml of phorbol 12‐myristate 13‐acetate (PMA) for 24 h in full RPMI medium.
9
Peripheral Blood Processing for Leukocyte Analysis
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On PD 130, a different batch of animals was decapitated, and the peripheral blood was collected in EDTA tubes. The blood was centrifuged at 500g for 5min at 4°C and the plasma was separated. 1.5 ml of blood was then transferred to a new tube and 10 ml of 1X lysis buffer (Biolegend) was added and allowed to react with the sample for 23 min. The samples were then centrifuged again at 500g for 5 min (4°C). Three washes with phosphate buffer saline (PBS) were performed and after the last wash, the pellet was resuspended in 400 μl of complete RPMI medium (Sigma-Aldrich Co). The entire procedure was carried out at 4°C.
10
Quantifying Antigen-Specific Plasma Cells
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For analysis of memory B-cells, splenocytes and lung cells were stimulated (5 × 10E5 cells per well, 24-well plate; 5 days, 37 °C) with 10 μg/mL CpG ODN 1826 (Invivogen, San Diego, CA), 10 μg/mL pokeweed mitogen (Sigma-Alderich, Zwijndrecht, The Netherlands), and Staphylococcus aureus protein A of Cowan Strain (1:5000; Sigma) in RPMI complete medium with β-mercaptoethanol (1:25000; Sigma) to induce differentiation into antibody secreting cells24 (link). The percentage of OMV specific antibody secreting cells were subsequently determined by ELISpot. The numbers of OMV-specific IgG- and IgA-producing plasma cells in blood, spleen and lungs were directly determined using the same ELISpot method, with 10 µg/mL wildtype B1917 OMV as coat, as described before15 (link). Spots were counted with an AID iSpot reader (Autoimmun Diagnostika, Strassberg, Germany) and indicated as antibody-secreting cells per 5 × 10E5 cells.
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