Cell scraper

RAW 264.7 cells were cultured in T-25 flasks in cell culture media consisting of DMEM and 10% fetal bovine serum. The cells were passaged every 3^rd day by resuspending the cells using a cell scraper (Fisher Scientific) and centrifuging the cells at 300XGs for 5 min. The supernatant was removed and the cells were then resuspended in cell culture media. The cells were counted using a hemocytometer (Fisher Scientific), and utilized for further experiments described below.

After incubation, treatments were removed and cells were washed with 2 mL of ice-cold 2× PBS. Caco-2 monolayers were lysed by adding 350 µL of mammalian protein extraction reagent/well (Thermo Fisher Scientific) (35 (link)) and incubated in 6-well plates for 10 min on a plate shaker at 120 rpm. Caco-2 monolayers were scraped with a cell scraper (Fisher Scientific), collected into microcentrifuge tubes, sonicated for 3 min, and centrifuged at 14,000 × g for 10 min at room temperature. Cell lysate supernatants were transferred to microcentrifuge tubes and stored at –20°C for ferritin and protein determination, which was completed within 24 h (21 , 35 (link), 36 (link)). Ten microliters of cell lysate solutions was used for determining ferritin concentrations (nanograms per milliliter) by using ELISA (Spectro Ferritin kit, S-22; Ramco Laboratories, Inc.), as done previously (23 (link), 36 (link)). Twenty-five microliters of cell lysate solutions was used for measuring protein concentrations with the use of Pierce bicinchoninic acid protein assay kits. Ferritin content (nanograms per milligram of cell protein) was calculated as a ratio of cell ferritin (nanograms per milliliter) to cell protein (milligrams per milliliter) (36 (link)).

After incubation, treatments were removed and cells were washed with 2 mL of ice-cold 2× PBS followed by 2 mL of ice-cold 2 g albumin/L in PBS. After discarding the washing solutions, Caco-2 monolayers were removed with the use of a cell scraper (Fisher Scientific) and collected into amber-colored microcentrifuge tubes using 1 mL ice-cold PBS. This process was repeated 2 additional times using 0.5 mL of ice-cold PBS, for a total of 2 mL PBS collected into the same microcentrifuge tubes and centrifuged at 327 × g for 45 min at 5°C. Supernatant PBS was discarded by carefully inverting the microcentrifuge tubes for 20 s. Tubes with cell pellets were then blanketed with nitrogen and stored at –80°C for vitamin A analysis (34 ).