Hadsell2 project protocol

Analysis of milk minerals in 31 inbred strains of mice   (2018)

Hadsell DL
With: Hadsell LA, Rijnkels M, Carcamo-Bahena Y, Wei J, Williamson P, Grusak MA

Project protocol - Contents

Workflow and sampling

Step Description Equipment Data collected
1 Individual matings set up between males and females within each strain (first pregnancy, first lactation) Breeding cages
2 Individual matings set up between males (CD-1) and females of each inbred strain (second pregnancy, second lactation) Breeding cages
3 Milk samples collected on day 9 postpartum from anesthetized dams and flash frozen Gentle vacuum, pre-weighed vials
4 Samples prepared and analyzed Inductively coupled plasma-optical emission spectroscopy Milk mineral concentrations


Procedure 1: Analysis of milk minerals

Equipment, software, and supplies

  • Vortex
  • Heat blocks
  • Inductively coupled plasma-optical emission spectroscopy (Spectro, CIROS ICP Model FCE12, Kleve, Germany)

Reagents and solutions

  • Nitric acid
  • Hydrogen peroxide
  • Tomato leaf standards (National Institute of Standards and Technology, SRM 1573A, Gaithersburg, MD, USA)

Environmental Conditions

Holding Area and/or Sample Collection Area

Anesthesia: Yes, not specified


  1. Study animals, animal husbandry, and experimental design are described in a previous publication (Hadsell et al. 2012).
  2. Milk is collected into pre-weighed vials from anesthetized dams (second lactation) at 9 days postpartum under a gentle vacuum and flash frozen.
  3. Milk samples are thawed at 37 °C and mixed by vortexing.
  4. A 0.2 mL sample is taken and weighed before transferring to a 100 mL glass digestion tube where samples are pre-digested overnight with 3 mL of ultra-pure concentrated nitric acid.
  5. Samples are heated to 125 °C on a digestion block for a minimum of 2.5h (with refluxing), until all the orange smoke dissipates from the tubes.
  6. Hydrogen peroxide (2 mL) is added to each tube and incubated at 125 °C for 1h. This step is repeated a second time.
  7. Samples are brought to dryness at 200 °C and then suspended in 10 mL of 2% nitric acid.
  8. Elemental analysis is carried out using inductively coupled plasma-optical emission spectroscopy. Certified standards are used to calibrate the instrument daily (tomato leaf standards).
  9. Elements are measured using the following emission lines: Ca (219.770 nm), Cu (327.390 nm), Fe (262.820 nm), K (766.490 nm), Mg (294.200 nm), Na (589.592 nm), P (253.565 nm), S (182.034 nm), and Zn (206.190 nm)


Primary References

Hadsell DL, Hadsell LA, Rijnkels M, Carcamo-Bahena Y, Wei J, Williamson P, Grusak MA. In silico mapping of quantitative trait loci (QTL) regulating the milk ionome in mice identifies a milk iron locus on chromosome 1. Mamm Genome. 2018 Oct;29(9-10):632-655. doi: 10.1007/s00335-018-9762-7. Epub 2018 Aug 2.   PubMed 30073618  

Protocol References

Hadsell DL, Wei J, Olea W, Hadsell LA, Renwick A, Thomson PC, Shariflou M, Williamson P. In silico QTL mapping of maternal nurturing ability with the mouse diversity panel. Physiol Genomics. 2012 Aug 17;44(16):787-98. doi: 10.1152/physiolgenomics.00159.201Epub 2012 Jul 3.   PubMed 22759921     FullText