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MB Sample ID: SA205461
| Local Sample ID: | 2bCre |
| Subject ID: | SU002225 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Male and female |
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Collection:
| Collection ID: | CO002218 |
| Collection Summary: | Both female and male mice were used in the experiments. Mice were maintained on a 12-h light/dark cycle with free access to food and water according to governmental rules. K320E-TWINKLE floxed mice (Baris et al., 2015) were crossed to CD23 CRE mice (Kwon et al., 2008) kindly provided by Meinrad Busslinger) to generate DNT animals. DNT mice used in these experiments had the genetic background DNT+/- CRE+/- and CRE control mice were DNT-/- CRE+/-. The WT animals used in this study were DNT-/- CRE-/- littermates. All mice were on the C57Bl/6 background. Isolation of primary murine cells from spleen and bone marrow Spleen was transferred into cold 2 % FCS (in PBS) and gently passed through a 70 µm cell strainer (BD) using the plunger of a 5 ml syringe (BD). Femur and tibia were flushed with cold 2 % FCS using a 27 G cannula (BD). Cell suspensions were pelleted by centrifugation at 300 x g for 5 min at 4°C. Erythrocytes were lysed in red blood cell-lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 100 µM EDTA) for 5min at room temperature. The reaction was stopped by adding cold 2% FCS before centrifugation at 300 x g for 5 min at 4°C. The final cell suspensions were kept in cold 2 % FCS after filtration through 30 µm mesh filter (Sysmex). In vitro cultivation of primary murine B cells Splenic B cells were cultured with a starting concentration of 0.5 x 106 cells/ ml in R10 medium (RPMI1640, 10 % fetal calf serum (FCS), 2 mM glutamate, 1 mM sodium pyruvate, 50 U/ml penicillin G, 50 μg/ml streptomycin, 50 μM β-mercaptoethanol) for 72 h at 37°C and 5% CO2, supplemented with 10 µg/ml LPS. For in vitro class switch recombination cells were seeded at 0.1 x 106 cells/ ml in R10 medium for 96 h, supplemented with 5 ng/ml transforming growth factor , 5 nM retinoic acid, 10 µg/ml anti-CD40 antibody, 10 µg/ml LPS, 100 U/ml IL4 and 10 ng/ml IL5. Ref.: Baris, O.R., Ederer, S., Neuhaus, J.F., von Kleist-Retzow, J.C., Wunderlich, C.M., Pal, M., WunderlichF.T., Peeva, V., Zsurka, G., Kunz, W.S., et al. (2015). Mosaic Deficiency in Mitochondrial Oxidative Metabolism Promotes Cardiac Arrhythmia during Aging. Cell Metab 21, 667–677. |
| Collection Protocol Filename: | Bcellscoll Mielenz.pdf |
| Sample Type: | B-cells |
