Cell Source
Normal human peripheral blood eosinophils are isolated from Buffy-coats by density centrifugation followed by negative selection using immune-magnetic anti-CD16 beads.
Cell Characterization
Eosinophils preparations were stained with May-Gruenwald-Giemsa. The purity of eosinophils is >=90%.
cDNA Prpeparation
One micrograms of total RNA is primed with random primer oligonucleotides and reverse-transcribed by MMLV reverse transcriptase in 20 µl final volume.
The RT reaction is stopped by heating at 65°C for 10 minutes.
The cDNA is delivered in 1x RT buffer. (1x RT Buffer: 50 mM Tris-HCl, pH 8.3, 75 mM KCI, 3 mM MgCI2, 10 mM DTT).
1 µl cDNA is sufficient for one PCR reaction.
The 5' end of human beta-actin cDNA has been amplified by PCR from all cDNAs.