Peripheral blood mononuclear cells (PBMCs) were prepared from heparinized blood by Ficoll-Hypaque density-gradient centrifugation. Isolated PBMCs were cultured in 24-well plates containing RPMI-1640 supplemented with 10% calf serum (Greiner, Wemmel, Belgium), 2 mM L-glutamine and 100 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). Cells were left unstimulated or were stimulated with a cocktail of anti-CD3 (5 µg/ml, eBioscience, San Diego, CA, USA) and anti-CD28 antibodies (5 µg/ml, eBioscience, San Diego, CA, USA) to mimic antigen presentation. Alternatively the cells were stimulated with LPS (1 ng/ml, Sigma-Aldrich Co. LLC), at 37°C in humidified 5% CO2 for 72 hours at a density of 1×106 cells/ml. Total RNA was extracted from the cultured PBMCs using TRIzol (Invitrogen), followed by reverse transcription using a transcriptase kit (Takara Biotechnology Co. Ltd., Dalian, China.). Real-time quantitative PCR was performed to compare the mRNA expression of TRAF3IP2 and TRAF5 gene, using the Applied Biosystem 7500 Real-time PCR System and was determined using the SYBR Green I Assay kit (Applied Biosysterms) and normalized to β-actin mRNA. Relative expression levels were calculated using the 2−ΔΔCt method. The sense and antisense primers used in this experiment are depicted in Table S4 .
Calf serum
Manufactured by Greiner
Sourced in Belgium, Germany
Calf serum is a cell culture medium supplement derived from the blood of calves. It provides a source of proteins, growth factors, and other nutrients essential for the growth and maintenance of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Applied biosystem 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
D8900, by Merck Group (1 mentions)
Holo transferrin, by Merck Group (1 mentions)
S5261, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
2 protocols using calf serum
1
PBMC Isolation and Gene Expression Analysis
2
Isolation and Culture of Luteal Stem Cells
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CLs classified in the mid luteal stage were collected for cell culture. Luteal tissue was enzymatically dissociated, and LSCs were cultured as described previously [45 (link)]. Dissociated LSCs from CLs were pooled and suspended in a culture medium, Dulbecco’s modified Eagle medium (DMEM) and Ham’s F-12 medium (D/F; 1:1 [vol/vol]; D8900; Sigma-Aldrich) containing 5% calf serum (16170–078; Life Technologies) and 20 μg/ml gentamicin (G1397; Sigma-Aldrich). Cell viability was greater than 85% as assessed by trypan blue exclusion. The cells in the cell suspension consisted of about 70% small LSCs, 20% large LSCs, 10% endothelial cells or fibrocytes, and no erythrocytes. Thus they mainly consisted of LSCs.
Dispersed LSCs (2.0 × 105 /ml) were cultured in D/F medium containing 5% calf serum in 10 cm2 culture dishes (664160; Greiner Bio-One, Frickenhausen, Germany) for determination of galectin-1 and VEGFR-2 protein expressions and in 96-well culture dishes (3860–096; Iwaki, Chiba, Japan) for determination of cell viability. After 24 h of culture, the medium was replaced with phenol red-free D/F medium (D2906; Sigma-Aldrich) containing 0.1% BSA, 5 ng/ml sodium selenite (S5261; Sigma-Aldrich), and 5 μg/ml holo-transferrin (T3400; Sigma-Aldrich), and the following experiments were carried out.
Dispersed LSCs (2.0 × 105 /ml) were cultured in D/F medium containing 5% calf serum in 10 cm2 culture dishes (664160; Greiner Bio-One, Frickenhausen, Germany) for determination of galectin-1 and VEGFR-2 protein expressions and in 96-well culture dishes (3860–096; Iwaki, Chiba, Japan) for determination of cell viability. After 24 h of culture, the medium was replaced with phenol red-free D/F medium (D2906; Sigma-Aldrich) containing 0.1% BSA, 5 ng/ml sodium selenite (S5261; Sigma-Aldrich), and 5 μg/ml holo-transferrin (T3400; Sigma-Aldrich), and the following experiments were carried out.
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