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HOME > Protocols > Cell Biology Protocols > Protocol for Assay of Myeloperoxidase in Frozen Lung Tissue

Protocol for Assay of Myeloperoxidase in Frozen Lung Tissue

  1. Thaw frozen lung samples and dice with razor blade.
  2. Homogenize lung tissue in 300ul working buffer.
  3. q.s. to 1ml with working buffer.
  4. Centrifuge at 10,000rpm in microfuge for 15 minutes.
  5. Discard supernatant.
  6. Resuspend pellet in 300ul working buffer with 50mM hexadecyltrimethylammonium bromide (HTAB).
  7. Homogenize for 30 seconds.
  8. Add 700ul of working buffer without HTAB and mix.
  9. Sonicate for 20 seconds.
  10. Snap freeze at -70oC or liquid nitrogen.
  11. Thaw to room temperature.
  12. Repeat steps 9-11 twice.
  13. Centrifuge at 10,000rpm for 10 minutes.
  14. Dilute aliquots 1:2 and 1:10 with to 200ul with working buffer with HTAB.
  15. Add 2.9 ml of working buffer containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide to 100ul of sample dilutions from step 14.
  16. Read absorbance at 460nm at regular intervals for 2 minutes (minimum 30 seconds).

Working buffer: 50mM potassium phosphate (pH 6.0)

The myeloperoxidase assay in mouse lung is used as a marker of airway inflamation due to asthma, environmental irratants, and repiratory infections.

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