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| === Permeabilization of C2C12 mouse myoblast cells with digitonin ===
| | [[Image:BB-Bioblast.jpg|left|40px|link=http://www.bioblast.at/index.php/Bioblast:About|Bioblast wiki]] |
| | | == Popular Bioblast page == |
| C2C12 mouse myoblast cells (0.5 million per ml)
| | ::: [[Permeabilized tissue or cells]] has been accessed more than |
| | | ::::* 5,000 times (2016-04-09) |
| Experimental buffer: MiR05
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| Rotenone: 1 µg/ml; Succinate: 10 mM; ADP: 2.5 mM; Digitonin: 5 mg/ml stock added in 1 µl steps up to 10 µg/ml
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| '''Experimental conditions:'''
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| 2 ml chamber volume, T = 37.0 °C
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| [[Image: C2C12 digitonin permeabilization.png|600px]] | |
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| '''Figure legends:'''
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| Digitonin concentration for permeabilization of cells must be optimized for each cell type/line and cell concentration used in respirometry. To this end, cells at the desired concentrations are introduced into the respiration chamber and the protocol shown here is applied. First, the ROUTINE rate of respiration observed in intact cells is determined. In this condition cells respire fuelled by endogenous substrates, since the respiration medium MiR05 does not contain any external substrates. Upon addition of complex I inhibitor rotenone respiration is blocked. Subsequently added substrates succinate and ADP do not stimulate respiration of intact cells, since there is no or only very limited uptake by the cells. The small increase of respiration may thus reflect the presence of a small percentage of damaged/dead cells. In the following, digitonin is added in small steps to progressively permeabilize the plasma membrane up to an optimum allowing unlimited access of substrates to the mitochondria, while leaving the mitochondrial membranes intact. Overtitration thus must be avoided as this will damage mitochondrial membranes and in consequence reduce respiration.
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| === Permeabilization of MG-63 human bone osteosarcoma cells with digitonin === | |
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| MG-63 human bone osteosarcoma cells (0.3 mio/ml)
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| Experimental buffer: MiR05
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| Rotenone: 1 µg/ml; Succinate: 10 mM; ADP: 2.5 mM; Digitonin: 5 mg/ml stock added in 1 and 2 µl steps up to 30 µg/ml
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| '''Experimental conditions:'''
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| 2 ml chamber volume, T = 37.0 °C
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| [[Image: MG-63 cells digitonin permeabilization.png|600px]] | |
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| '''Figure legends:'''
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| Optimization of digitonin concentration for permeabilization of MG-63 cells, following the protocol described above for C2C12 cells.
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