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Protocol
for Assay of Myeloperoxidase in Frozen Lung Tissue
- Thaw frozen lung samples and dice with razor blade.
- Homogenize lung tissue in 300ul working buffer.
- q.s. to 1ml with working buffer.
- Centrifuge at 10,000rpm in microfuge for 15 minutes.
- Discard supernatant.
- Resuspend pellet in 300ul working buffer with 50mM
hexadecyltrimethylammonium bromide (HTAB).
- Homogenize for 30 seconds.
- Add 700ul of working buffer without HTAB and mix.
- Sonicate for 20 seconds.
- Snap freeze at -70oC or liquid nitrogen.
- Thaw to room temperature.
- Repeat steps 9-11 twice.
- Centrifuge at 10,000rpm for 10 minutes.
- Dilute aliquots 1:2 and 1:10 with to 200ul with
working buffer with HTAB.
- 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.
- 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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