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To quantify this difference, the three biggest PPs of each mouse were pooled and their cells were counted.
With the exception of the analysis conducted for each muscle group, skeletal muscles from each mouse were pooled and treated as individual samples.
Homogenates from both legs of the same mouse were pooled and passed though a 100 µm cell strainer (BD Falcon) by centrifugation at 1100×g for 5 minutes at 4°C to filter residual myofibrillar material from the nuclei.
The serial lavage aliquots recovered from a single mouse were pooled (∼4 ml) and mixed with an equal volume of complete RPMI medium (RPMI 1640, 10% FBS, 2% penicillin-streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM non-essential amino acids, 50 µM β-mercaptoethanol).
The vaginas of 2 3 mice per time point were washed 3 times with 40 µL PBS and the three samples from each mouse were pooled (∼120 µL) and centrifuged at 13,000 rpm for 3 min. Supernatants were frozen at −20°C.
For PND22 pups and adults, both oviducts from one mouse were pooled per biological replicate.
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The set of inguinal lymph nodes from each mouse was pooled and used in each assay.
Mouse blood (200 900 μl per mouse) was pooled and collected into a 2-ml EDTA tube (BD, Franklin Lakes, NJ, USA).
For SAGE analysis, in both treated and control groups, an equal amount of kidneys extracted RNAs from each mouse was pooled into a single sample.
Once the mice were 10 weeks of age, spot urine samples were collected daily in the morning for 1 week, and the urine for each mouse was pooled and centrifuged for 5 min at 10,000 rpm.
Urine samples collected from Ate1-deficient mice were pooled and compared with pooled urine from Ate1-containing mice.
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