Primary B cells were isolated from mouse spleens by immunomagnetic depletion with anti-CD43 MicroBeads (Miltenyi Biotech) [45] (link). The harvested cells were stimulated in the presence of 25 μg/mL LPS (Roche) and 50 ng/mL IL-4 (Sigma-Aldrich) for three days. After the stimulation, RNA was isolated for a further analysis.
Lipopolysaccharides (lps)
Manufactured by Roche
Sourced in Germany
The LPS is a laboratory equipment product designed for use in various research and diagnostic applications. It serves as a core component in laboratory workflows, providing essential functionality without interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Merck Group (2 mentions)
Anti cd43 microbeads, by Miltenyi Biotec (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lps escherichia coli o55 b5, by Merck Group (1 mentions)
Htb 38, by American Type Culture Collection (1 mentions)
Pegifnα 2a, by Roche (1 mentions)
5 protocols using lipopolysaccharides (lps)
1
Isolating and Stimulating Primary B Cells
2
Reprogramming Mouse Primary B Cells
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CD43-negative primary B cells were isolated from mouse spleens and stimulated in the presence of 25 μg/mL LPS (Roche) and 50 ng/mL IL-4 (Sigma-Aldrich) for 24 h. Then, 106 cells were cultured in 2 mL of the medium containing retroviruses encoding reprogramming factors with LPS and IL-4 in one well of a six-well plate for two days. After the infection, the medium was changed to ES medium, and 5×105 cells were reseeded onto SNL feeder cells in the 100 mm dishes. On days five and seven, 10 mL of ES medium was added to the dish, and the medium was replaced on day nine. Twenty-five days after the B cell isolation, the number of GFP-positive colonies was counted.
3
Isolation and Culture of Peritoneal Derived Macrophages
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PDMs were isolated from 50 male C57BL/6 mice (6-8 weeks old, 20-25 g), purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The C57BL/6 mice were housed under controlled conditions at a temperature of 25±2˚C with a 12-h light-dark cycle and 55±2% relative humidity, as previously described (32 (link)). Mice were and sacrificed by intraperitoneal injection of overdoses of sodium pentobarbital (150 mg/kg). PDMs were cultured in an RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere under 5% CO2 and 95% O2 at 37˚C. Cells were randomly separated into three groups: Normal (N), LPS+ATP and AKFPD groups. PDMs in the LPS+ATP group were stimulated with 500 ng/ml lipopolysaccharide (LPS; cat. no. L8880; Beijing Solarbio Science & Technology Co., Ltd.) for 2.5 h, followed by exposure to 5 mM ATP (cat. no. 10519979001; Roche Diagnostics) for 0.5 h at 37˚C. PDMs in the AKFPD group were pre-incubated with 400 µg/ml AKFPD for 24 h at 37˚C and subsequently exposed to LPS plus ATP.
4
HT-29 Cells: IFN-γ and LPS Responses
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The human intestinal epithelial cell lines HT-29 (Organism Homo sapiens, human/tissue colon/disease: colorectal adenocarcinoma) was purchased by ATCC®HTB-38™ [17 (link)] were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Life Technologies), 2 mM glutamine, penicillin (25 U/mL) and streptomycin (25 mg/mL) in a 5% CO2 atmosphere at 37°C. Cells were pretreated with Human interferon-γ (IFN-γ) (Roche Applied Science, Germany) 10 ng/ml for 12 hours or control medium as previously described in [21 (link)], washed, and then stimulated with LPS 50 ng/ml at different times. LPS (Escherichia coli, O55:B5) were purchased from Sigma-Aldrich (St. Louis, MO) and reconstituted in endotoxin-free water.
5
Stimulation of Cultured FDCs and T-B Cell Interactions
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Cultured FDCs were stimulated with IL-4 (30 ng/mL), IL-10 (30 ng/mL), IL-21 (30 ng/mL), lipopolysaccharide (LPS, 1 μg/mL), Peg-IFNα-2a (2 μg/mL, Roche, Shanghai, China), lymphotoxin-α1β2 (10 ng/mL), tumor necrosis factor (TNF)-α (10 ng/mL), CPG (5 μg/mL), or PMA (50 ng/mL) for 3 days, respectively. Then, the supernatants were collected, and the levels of IL-6 and CXCL13 were assessed by ELISA. Autologous CD4+ T cells and CD19+ B cells were sorted from SMCs of patients with chronic HBV infection by FACS and cryopreserved in liquid nitrogen. Purified autologous CD4+ T cells were thawed and plated in a 96-well plate that was preseeded with FDCs at an 80:1 ratio (CD4+ T cells/FDCs), or with medium only as a control, and co-cultured in the presence of IL-2 (10 ng/mL) and anti-CD3/CD28 (10 μg/mL) for 3 days, the supernatants were collected, and the levels of IL-21 and IFN-γ were assessed by ELISA.
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