Intracellular total Iron levels were measured using Iron Assay Kit from Abcam. Briefly, H727 cells were transiently transfected with pCS2-HBB plasmids for 24 h, and then provided with fresh medium containing 10 μM (Fe2+) for 8 h. Cell lysates were then collected and intracellular total Iron levels were measured using the Iron Assay Kit (Abcam) according to manufacture's protocols.
Iron assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom, China
The Iron Assay Kit is a laboratory product designed to quantify the iron content in a variety of sample types, including biological samples. The kit provides the necessary reagents and protocols to perform this analysis accurately and efficiently.
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241 protocols using iron assay kit
1
Measuring Intracellular Iron Levels
2
Quantification and Visualization of Cellular Iron
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Iron concentration was measured using either QuantichromeTM Iron Assay Kit (BioAssay System, 18614-83) or Iron Assay Kit (Abcam, ab83366) following manufacturers’ instruction87 (link). For measuring intracellular iron, cell pellets were harvested after pre-incubation with ferric ions and washed twice with cold phosphate-buffered saline (PBS), followed by lysis in RIPA buffer. Then, iron levels were determined by the absorbance at 590 nm after incubation in iron detection reagent. Absorbance of a sample blank -supernatant without iron detection reagent- was subtracted to determine supernatant iron. The Abcam Iron Assay Kit was used for determining total iron, ferrous ions, and ferric ions by adding or omitting the iron reducer. After pre-incubating with ferric ions and dopamine, cell lysates were prepared by iron assay buffer provided in the Kit to measure iron as instructed in the protocol. The iron level was normalized by protein concentration. For tissue iron staining, Iron Stain Kit (Prussian Blue Stain) (Abcam, ab150674) was used by following manufacturer’s procedure. Frozen tissues were washed with distilled water and stained with iron stain solution for 3 min. Tissue morphology was visualized by provided nuclear fast red solution.
3
Quantification of Cellular Iron Levels
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We detected the concentration of cellular irons according to the manufacturer’s introductions of iron assay kit (Abcam, United States). After treated with ferroptosis inducer erastin (5 μM) or RSL3 (1 μM) for 24 h, cells were rapidly mixed with iron assay buffer. We then removed the insoluble material, and added the iron assay buffer and iron probe into the reaction mixture. At last, the spectrometric absorbance was detected at the wavelength of 593 nm.
4
Extraction and Analysis of Wheat Apoplastic Fluids
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Extraction of apoplastic fluids was performed by using a previously described method with slight modifications (39 (link)). Briefly, 14-day-old wheat seedling leaves inoculated with Fg at different time intervals were washed and vacuum infiltrated with cold water twice for 2 min. Apoplastic fluids were collected by centrifugation at 3000 × g for 5 min, and then were used for further iron content and pH measurement.
Iron content of apoplastic fluids from Fg infected wheat seedling leaves were assayed by using an Iron Assay Kit (Abcam, ab83366), and Fe2+ content was measured by using the Synergy H1 microplate reader (OD = 593 nm; Biotek Instruments, Winooski, VT, USA) following the manufacturer's instructions (40 (link)). The pH of apoplastic fluids from wheat seedling leaves and palea tissues sampled at different time intervals after Fg inoculation was measured with a pH electrode (model PHM93, Radiometer Analytical).
Iron content of apoplastic fluids from Fg infected wheat seedling leaves were assayed by using an Iron Assay Kit (Abcam, ab83366), and Fe2+ content was measured by using the Synergy H1 microplate reader (OD = 593 nm; Biotek Instruments, Winooski, VT, USA) following the manufacturer's instructions (40 (link)). The pH of apoplastic fluids from wheat seedling leaves and palea tissues sampled at different time intervals after Fg inoculation was measured with a pH electrode (model PHM93, Radiometer Analytical).
5
Quantitative Iron Assay Protocol
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The levels of Fe2+ and total iron were determined using the iron assay kit (Abcam, Cambridge, MA), following the manufacturer’s instructions.
6
Neferine Modulates Iron Levels
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IHH-4 and CAL-62 cells with an inoculation density of 1 × 105 cells/well were seeded into 96-well plates and incubated for 24 h at 37°C with 5% CO2. Subsequently, both cells were introduced with 5 μM neferine, 10 μM neferine, 5 μM Ferrostatin-1 (Fer-1, the inhibitor of ferroptosis), 5 μM neferine plus 5 μM Fer-1, and 10 μM neferine plus 5 μM Fer-1 for 24 h. The relative iron level of cells was measured by an iron assay kit (Abcam, Cambridge, UK) in line with the handling instruction.
7
Cellular Iron Concentration Quantification
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According to the manufacturer's instruction [26 (link)], Iron Assay Kit (ab83366, Abcam) was used to measure cellular iron concentration. In brief, 5 × 106 cells were seeded on to the plate and then pretreated with drugs for 24 h. Before centrifuging (13,000×g, 10 min) at 4 °C, cells were homogenized by 5 × volumes of iron assay buffer. Next, iron reducer and supernatant mixture were incubated at room temperature for 30 min. Then, each sample was added iron probe and incubated without light on the horizontal shaker for 60 min. The absorbance was detected at 593 nm by EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).
8
Quantifying Cellular Iron Levels
9
Intracellular Iron and Lipid ROS Detection
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The relative level of intracellular iron was detected by an Iron Assay Kit (Abcam, USA) following manufacturer’s protocol. The lipid ROS was detected by using C11-BODIPY (Invitrogen) staining. In brief, cells were planted on 12-well plates and incubated overnight. After indicated incubation, cells were collected and resuspended in 200 μL PBS, stained with C11-BODIPY (5 μM) at 37°C for 30 minutes. Samples were examined by flow cytometer (BD, USA). The level of malondialdehyde (MDA) was assessed by using a lipid peroxidation (MDA) assay kit (Sigma). Cells were lysed by MDA lysis buffer on ice, followed by centrifugation at 13,000 × g for 5 minutes. The absorbance values were detected at 532 nm by a spectrophotometer (BD Biosciences).
10
Lipid ROS and Iron Quantification
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The accumulated lipid ROS and iron level were measured by C11-BODIPY (Thermo, USA) and iron assay kit (Abcam, USA) as per manufacturers' protocols.
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