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The amount of spores that needs to be added to samples to obtain suitable Cq values for this internal control must be determined empirically for each stock spore suspension.
In brief, Coons found that if a fluorescent molecule were chemically hooked to purified antibody, this labeled antibody could be added to samples of tissue to find the suspected antigen (15).
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For the lipid extraction process, n-hexane solution (Sigma-Aldrich) was added to samples in 6 1 ratio (v/w).
Two hundred (200) microliters of HCl in methanol (2%) was added to samples before drying with a Turbovap N2 evaporator.
Briefly, an internal standard (in-house compound) was added to samples followed by protein precipitation involving the addition of acetonitrile.
7AAD was added to samples to exclude dead cells from sorts.
After the final wash, 1 µg/ml of propidium iodide was added to samples to eliminate dead cells.
1×104 splenocytes loaded with Indo-1 (Invitrogen, Carlsbad, CA) were added to samples and analyzed by FACS.
Orange loading dye was added to samples which were loaded on pre-run 5% TBE gels and run at 70 V for 2 h.
EDTA was added to samples immediately before staining.
EDTA (1 mM) was added to samples without FeSO4.
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