Potter—Elvehjem homogenizer

Transfer the dissected brain(s) to a prechilled glass Potter-Elvehjem homogenizer containing ice-cold isotonic sucrose solution. Use a 1 10 w/v ratio of tissue to sucrose solution (see Notes 5 and 6). 

Add 5 mL of ice-cold Milli-Q or double-distilled water to the P2 fraction, resuspend it, and pipet it into a prechilled Potter-Elvehjem homogenizer. Rinse the centrifuge tube with 1 mL of ice-cold water, giving a final volume of approx 7 mL m the homogenizer. Manually homogenize the P2 pellet using 5 up-and-down strokes. 

Discard the supernatant and resuspend the resulting SPM pellet in a volume of ice-cold SPM buffer sufficient to make an approx S mg protein/mL solution (see Note 14) and transfer the suspension to a 1 mL Potter-Elvehjem homogenizer. Homogenize the suspension manually, on ice, using 5 up-and-down strokes. 

Mouse livers were excised and homogenized with a Potter-Elvehjem homogenizer. The microsomal fraction was isolated by centrifuging the homogenate at 9000 x g for 15 min, to sediment nuclei, mitochondria, and membrane debris. The supernatant was centrifuged at 100,000 x g for 60 min to pellet microsomes. The microsomes were further purified by another centrifugation step to remove soluble proteins (e.g., hemoglobin). HCQ was incubated with the microsomes for 2 h. Then a liquid-liquid extraction technique was performed to recover HCQ and its metabolites. The molecules were separated by a commercial CE system with ultraviolet (UV) absorbance detection. 

When Ca " " was omitted 0.2 mM EGTA was added. Thereafter at different time intervals the retinas were removed from the incubation media and homogenized with chloroform-methanol by means of a Potter-Elvehjem homogenizer (Folch et al., 1957). Phospholipid classes were isolated by two-dimensional TLC (Rouser et al., 1970) and neutral glycerides by gradient-thickness TLC (Bazan Bazan, 1975). 

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