The immortalized mouse cardiac endothelial cell line (MMEC) was cultured in DMEM supplemented with 5% FBS (GIBCO) on collagen. Cells were incubated overnight in 0% FBS and stimulated with 300 ng/ml LPS for 4 h with or without preincubation for 60 min with a Map3k8 inhibitor (C1, 10 μM) that was generously provided by Sir Philip Cohen, and G-CSF levels in the cell supernatant were determined. The human endothelial cell line HMEC-1 was cultured in MCDB131 medium (Corning) supplemented with 10% FBS (GIBCO), 10 ng/ml EGF (E4127, Sigma-Aldrich), 1 µg/ml hydrocortisone (H0888, Sigma-Aldrich), 10 mM glutamine (G7513, Sigma-Aldrich), 100 U/ml penicillin (Gibco, Thermo Fisher Scientific) and 100 µg/ml streptomycin (Gibco, Thermo Fisher Scientific). Cells were incubated overnight in 0.1% FBS and stimulated with 300 ng/ml LPS for 6 h with or without preincubation for 60 min with the Map3k8 inhibitor. The cells were then harvested and subjected to quantitative reverse-transcriptase PCR analysis.
Hydrocortisone h0888
Manufactured by Merck Group
Sourced in New Zealand
Hydrocortisone (H0888) is a laboratory reagent manufactured by Merck Group. It is a glucocorticoid hormone used as a research tool in various applications. The product specifications and details are available on the Merck website.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Mcdb131 medium, by Corning (1 mentions)
Egf e4127, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Crl 5822, by American Type Culture Collection (1 mentions)
E9644, by Merck Group (1 mentions)
C8052, by Merck Group (1 mentions)
Clls1042, by Merck Group (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Indirect cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Ms macs separation column, by Miltenyi Biotec (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Myelocult h5100, by STEMCELL (1 mentions)
3 protocols using hydrocortisone h0888
1
Immortalized Endothelial Cells Response to LPS
2
Isogenic Cell Lines for Cancer Research
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MCF10A, a non-tumorigenic breast cell line (ATCC, CRL-10317) and MCF10A CDH1−/− cells (CLLS1042, Sigma-Aldrich, St Louis, MO, USA) were cultured in DMEM/F12/Glutamax (10565042, Thermo Fisher Scientific, Waltham, MA, USA) with 5% horse serum (16050, Thermo Fisher Scientific, Waltham, MA, USA), 10μg/μL neutral insulin (797139, Dunedin hospital, Dunedin, New Zealand), 500 ng/mL hydrocortisone (H0888, Sigma-Aldrich, St Louis, MO, USA), 100 ng/mL cholera toxin (C8052, Sigma-Aldrich, St Louis, MO, USA) and 20 ng/mL human epidermal growth factor (E9644, Sigma-Aldrich, St Louis, MO, USA). A CDH1-null strain of the gastric cancer cell line NCI-N87 (ATCC, CRL-5822) was generated using CRISPR-Cas9 [9 (link)]. Briefly, the sequence 5′-ATTCACATCCAGCACATCCA-3′ was used to target exon 10 of CDH1 and induce a single base frameshift, followed by a stop codon. Isogenic NCI-N87 cells were cultured in DMEM/F12/Glutamax with 10% fetal bovine serum (10091148, Thermo Fisher Scientific, Waltham, MA, USA). Both cell lines were grown at 37 °C with 5% CO2.
3
Hematopoietic Support by iPSC-Derived MSCs
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MSC-like cells generated from hiPSC were seeded at 30,000 cells/cm2 in 96-well plate (n = 5) onto 0.1% gelatine-coated wells in MSC medium. At 100% confluence, the cells were irradiated at 10 Gy for 10 min. The same day, CD34+ hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10−6 hydrocortisone (H0888; Sigma-Aldrich). Co-cultures were maintained for 5 weeks with medium changes every other day and then evaluated by optical microscopy analysis on a Nikon Eclipse Ti microscope (Nikon) for the formation of typical cobblestone areas and by flow cytometry using CD45-APC—C7 (561,863; BD; RRID: AB_10,897,014) and CD34-FITC antibodies (555,821; BD; RRID: AB_396,150) to assess support of hematopoietic cells, following the protocols already described above.
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