TER was measured using Cellzscope (nanoAnalytics, Münster, Germany). MDCK-II cells were seeded on hanging PET cell culture inserts (Merck Millipore, #PIHT12R48) at a density of 80,000 cells/insert. After TER values had reached a stable plateau, the cells were transferred to a 24-well plate and washed twice with HBSS+/+ warmed to 37°C. Lucifer yellow (m.w. 457 Da; Sigma-Aldrich, #L0259) was dissolved in warm HBSS+/+ (200 μM) and 200 μl solution were applied to the apical compartment of the insert, in order to determine the permeability coefficient of the cell barrier [23 (link)]. The basolateral compartment was filled with 1ml warm HBSS+/+ and the plate was incubated at 37% with 10% CO2 for 10 min. The inserts were then moved to a fresh 24-well plate containing prewarmed HBSS+/+ and incubated for two more 10 min periods. From each basolateral compartment, 80 μl of sample were transferred to a 96-well plate in triplicate and Lucifer yellow fluorescence was measured using a Tecan microplate reader (Tecan, Männedorf, Switzerland); concentrations were determined from a standard curve.
Piht12r48
Manufactured by Merck Group
PIHT12R48 is a precision incubator from Merck Group. It is designed for temperature-controlled incubation of samples or cultures. The device maintains a stable temperature within the specified range to support various laboratory applications.
Automatically generated - may contain errors
2 protocols using piht12r48
1
Measuring Epithelial Permeability via Lucifer Yellow
2
Investigating PLAC8 Impact on CRC
Check if the same lab product or an alternative is used in the 5 most similar protocols
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overPLAC8-SW480 cells were used to assess the effect of PLAC8 on the growth of CRC cells. The relative growth rate was determined by counting cells after different incubation intervals using a Scepter Handheld Automated Cell Counter (Merck KGaA, Darmstadt, Germany). Briefly, cells were counted after different incubation intervals (24, 48, 72 h), and the cell growth rates were expressed relative to the number at the initial seeding. A cell migration experiment with CRC cells that did or did not overexpress PLAC8 was conducted using a polyethylene terephthalate hanging Transwell insert (diameter, 8 mm) with a pore size of 0.4 μm (PIHT12R48; Merck KGaA) according to our previous publication with minor modifications [13] (link). Briefly, the times for crystal violet staining were 30 and 60 min for SW480 cells and 20 and 40 min for SW620 cells. The numbers of migrating cells reported here represent the average value ± standard deviation obtained from 2 to 3 independent experiments.
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