2017年5月24日(水)
NP dilution with cell growth
NP dilution with cell growth was monitored over 6 days by ICP-MS by the quantification of intracellular levels of Zn. NB4 and THP-1 cells (0.5 × 106 cells per ml) were plated in six-well plates and incubated in serum-free RPMI-1640 with 20?μg?ml?1 of RA+NPs. After 4?h incubation, NPs that were not internalized by the cells were washed three times with PBS and the cells were left to grow at 0.2 × 106 cells per ml in complete medium for additional 4?h, 3 days and 6 days, maintaining always an exponential growth. After each incubation, cells were counted, collected by centrifugation and resuspended in nitric acid (1?ml, 69% (v/v)) for ICP analysis. The concentration of Zn was normalized per cell. The estimation of NPs was done based on Zn quantification in 20?μg of NPs. In some experiments, cells were transfected with RA+NPs labelled with TRITC, and their fluorescence monitored by flow cytometry overtime, to evaluate NPs distribution within the cells.
[3H]RA internalization studies
[11, 12-3H(N)]-Retinoic acid, 50.4?Ci?mmol?1, was purchased from Perkin Elmer. [3H]RA solution for cell culture assays was prepared on the day of experiments by dissolving [3H]RA in DMSO with unlabelled RA in a 1:1,000 ratio to a final concentration of 10?μM of RA. [3H]RA solution in DMSO for the preparation of NPs was prepared on the day of experiments using a 1:4,000 ratio of labelled to unlabelled RA. Experiments were initiated by the adding the [3H]RA solution (1?μM and 10?μM; representing less than 1% in volume of the total cell culture medium) or [3H]RA-NP suspension (1?μg?ml?1 and 10?μg?ml?1) to cultures (60,000 cells per condition, 24-well plate, 1?ml) of NB4 or U937 cells. In case of soluble RA, cells (NB4 or U937; 60,000 cells per condition, 24-well plate) were cultured with medium containing [3H]RA (1?μM and 10?μM; 1?ml of medium) for 24 or 72?h, washed with PBS (two times), collected, lysed with lysis buffer (100?ml) and kept on ice until scintillation counting procedure. In case of RA-containing NPs, cells (same conditions as for soluble RA) were cultured with [3H]RA-NPs (1?μg?ml?1 and 10?μg?ml?1) for 4?h, washed with PBS and cultured for additional 20 or 68?h in the respective culture medium. Cells were then collected to eppendorfs, washed with PBS, centrifuged (1,500?r.p.m., 5?min), lysed with lysis buffer (see above) and kept on ice until scintillation CATV splitter counting procedure. The lysed samples (100?ml) were mixed with liquid scintillation fluid (1?ml; Packard Ultima Gold) and the scintillations counted in a TriCarb 2900 TR Scintillation analyser (Perkin Elmer).
NB4 differentiation assay
[3H]RA internalization studies
[11, 12-3H(N)]-Retinoic acid, 50.4?Ci?mmol?1, was purchased from Perkin Elmer. [3H]RA solution for cell culture assays was prepared on the day of experiments by dissolving [3H]RA in DMSO with unlabelled RA in a 1:1,000 ratio to a final concentration of 10?μM of RA. [3H]RA solution in DMSO for the preparation of NPs was prepared on the day of experiments using a 1:4,000 ratio of labelled to unlabelled RA. Experiments were initiated by the adding the [3H]RA solution (1?μM and 10?μM; representing less than 1% in volume of the total cell culture medium) or [3H]RA-NP suspension (1?μg?ml?1 and 10?μg?ml?1) to cultures (60,000 cells per condition, 24-well plate, 1?ml) of NB4 or U937 cells. In case of soluble RA, cells (NB4 or U937; 60,000 cells per condition, 24-well plate) were cultured with medium containing [3H]RA (1?μM and 10?μM; 1?ml of medium) for 24 or 72?h, washed with PBS (two times), collected, lysed with lysis buffer (100?ml) and kept on ice until scintillation counting procedure. In case of RA-containing NPs, cells (same conditions as for soluble RA) were cultured with [3H]RA-NPs (1?μg?ml?1 and 10?μg?ml?1) for 4?h, washed with PBS and cultured for additional 20 or 68?h in the respective culture medium. Cells were then collected to eppendorfs, washed with PBS, centrifuged (1,500?r.p.m., 5?min), lysed with lysis buffer (see above) and kept on ice until scintillation CATV splitter counting procedure. The lysed samples (100?ml) were mixed with liquid scintillation fluid (1?ml; Packard Ultima Gold) and the scintillations counted in a TriCarb 2900 TR Scintillation analyser (Perkin Elmer).
NB4 differentiation assay
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