For cell culture experiments, we used mesenchymal stem cells (hMSC): bone marrow-derived (female, 26 years) and adipose-derived (female, 18-30 years) and THP-1 cells, purchased from RoosterBio and American Type Culture Collection (ATCC), respectively. The hMSC cells were maintained in DMEM high glucose medium with L-glutamine (D5796, Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (F6765, Sigma-Aldrich), 1% penicillin–streptomycin (P4333, Sigma-Aldrich), and 1% NEAA (M7145, Sigma Aldrich). The THP-1 cells were maintained in RPMI-1640 medium (R7388, Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (F6765, Sigma-Aldrich), 1% penicillin–streptomycin (P4333, Sigma-Aldrich), and 0.05 mM 2-mercaptoethanol. All cells were cultured at 37 °C in 5% CO2 and were mycoplasma-free. The hMSC passages were performed at 75-80% confluence using 0.25% trypsin (59418C, Sigma-Aldrich). The hMSCs were used below seven passages, and the THP-1 cells were used below 40 passages. Viable cells were counted using a hemocytometer and the trypan blue exclusion method (T8154, Sigma–Aldrich). The differentiation of THP-1 monocytes to macrophages occurred after treatment with 18 nM of phorbol 12-myristate 13-acetate (1585, Sigma-Aldrich) for 12 hrs. Macrophage M2 polarization was performed using 20 ng/mL of IL-13 and 20 ng/mL of IL-4 for 72 hrs.
R7388
Manufactured by Merck Group
The R7388 is a laboratory equipment product manufactured by Merck Group. It serves as a core function in laboratory settings, but a detailed description cannot be provided while maintaining an unbiased and factual approach. More information may be available from the product's documentation or by contacting Merck Group directly.
Automatically generated - may contain errors
3 protocols using r7388
1
Mesenchymal Stem Cell and Macrophage Differentiation
2
Mesenchymal Stem Cell and Macrophage Differentiation
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For cell culture experiments, we used mesenchymal stem cells (hMSC): bone marrow-derived (female, 26 years) and adipose-derived (female, 18-30 years) and THP-1 cells, purchased from RoosterBio and American Type Culture Collection (ATCC), respectively. The hMSC cells were maintained in DMEM high glucose medium with L-glutamine (D5796, Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (F6765, Sigma-Aldrich), 1% penicillin–streptomycin (P4333, Sigma-Aldrich), and 1% NEAA (M7145, Sigma Aldrich). The THP-1 cells were maintained in RPMI-1640 medium (R7388, Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (F6765, Sigma-Aldrich), 1% penicillin–streptomycin (P4333, Sigma-Aldrich), and 0.05 mM 2-mercaptoethanol. All cells were cultured at 37 °C in 5% CO2 and were mycoplasma-free. The hMSC passages were performed at 75-80% confluence using 0.25% trypsin (59418C, Sigma-Aldrich). The hMSCs were used below seven passages, and the THP-1 cells were used below 40 passages. Viable cells were counted using a hemocytometer and the trypan blue exclusion method (T8154, Sigma–Aldrich). The differentiation of THP-1 monocytes to macrophages occurred after treatment with 18 nM of phorbol 12-myristate 13-acetate (1585, Sigma-Aldrich) for 12 hrs. Macrophage M2 polarization was performed using 20 ng/mL of IL-13 and 20 ng/mL of IL-4 for 72 hrs.
3
Koala Leukocyte MOMP Stimulation
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Three millilitres RPMI medium (Sigma-Aldrich #R7388), was incubated for 30 min at 37 °C. The same volume of heparanised blood from each koala was centrifuged at 3000 g for 5 min and the leukocyte fraction (buffy coat) was aspirated and suspended in the pre-incubated media. In duplicate, 5 µg of rMOMP genotype G in 25 µl of foetal calf serum (FCS) or, in the case of negative controls FCS 25 µl, was added to 220 µl of the buffy coat solution. Suspensions were then incubated for 12 h at 37 °C then 750 µl of RNA later (Sigma-Aldrich #R0901) was added to each sample and the sample stored at room temperature for 24 h, then frozen at − 20 °C until processing.
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