Blood was collected by submandibular bleeding and analyzed for CBC using Heska HT5 Element analyzer at the University of Michigan In-Vivo Animal Core (Ann Arbor, MI) on a fee-for-service basis. Tissue non-heme iron contents were measured as previously reported (74 (link)). Briefly, tissues were homogenized in a buffer containing 1 M HCl and 10% (wt/vol) trichloroacetic acid and heated for 1 h at 95 °C. Following a high-speed spin, iron was quantitated using a ferrozine solution and compared with a standard. Serum iron, ferritin, and hepcidin concentrations were measured using the Serum Iron Assay Kit (Abcam ab239715), Mouse Ferritin ELISA Kit (Abclonal RK02793), and Hepcidin Murine-Compet ELISA Kit (Intrinsic Lifesciences HMC-001), respectively, according to the manufacturer’s instructions. Copper content in tissues and serum was measured using a Copper Assay Kit (Sigma MAK127) according to the manufacturer’s instructions. Serum CP activity was measured using a Ceruloplasmin Colorimetric Activity Kit (Arbor Assay K035-H1) according to the manufacturer’s instructions.
Serum iron assay kit
Manufactured by Abcam
Sourced in United States
The Serum Iron Assay Kit is a laboratory tool designed to measure the concentration of iron in serum or plasma samples. It provides a quantitative assessment of the iron levels present in the tested biological samples.
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4 protocols using serum iron assay kit
1
Comprehensive Blood and Tissue Iron Analysis
2
Quantification of Iron Status
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Whole blood was used immediately to measure haemoglobin (haemocue Hb 201+) concentrations and haematocrit. Serum iron was determined using a commercial kit based on the total iron-binding capacity and serum iron assay kit (Abcam, Waltham, MA, USA). Tissue iron content was measured to assess iron stores using a standard ferrozine assay used in a previous study.(35 (link)) Briefly, livers were homogenised in water and subjected to trichloroacetic acid (TCA) protein precipitation at 65 °C for 20 h. Non-haeme iron assay was determined calorimetrically using ferrozine in thioglycolic acid by measuring the absorbance using a microplate reader (BioTek Instruments, Winooski, VT, USA) to assess soluble iron and calculating tissue iron content based on the weight of the tissue used. The same process was used to measure spleen iron content.
3
Quantifying Iron Homeostasis in Mtb Infection
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Total iron-binding capacity (TIBC), total iron, and percentage transferrin saturation (%Tf) in plasma and lung homogenates of uninfected or Mtb CDC1551-infected rabbits with or without Fe-treatment was determined using a colorimetric assay (TIBC and Serum Iron Assay Kit) following the manufacturer’s instructions (BioVision, Milpitas, CA, USA). This experiment was performed in duplicate with samples from three animals per time point per condition.
4
Iron-bound Transferrin Measurement in Mice
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For iron-bound transferrin measurement, mouse blood was collected from the heart of mice sacrificed at the end of second FMD cycle and 24 h post-refeeding. Blood was incubated at room temperature (25 °C) for at least 30 min to clot and then centrifuged for 15 min at 2000 × g (4 °C). Collected serum was aliquoted and stored at −80 °C. Iron-bound transferrin was measured by Serum Iron Assay kit (Biovision, #K392) according to manufacturer protocol.
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