PI staining of PBLs
Manabu Takahashi (manabu32@ymg.urban.or.jp)
Mon, 16 Dec 1996 00:02:33 +0900 (JST)
Dr. Ormerod wrote :
>The cells were then washed and fixed in 70% ethanol on ice. After 1 to 24
>hours, the cells were washed and resuspendedin a buffer containing PI and
>RNase and incubated at 37 for 30 min.
>We consistently obtain two peaks in the DNA histogram (30% difference).
>If we use a detergent method to prepare unfixed nuclei, there is only one peak
>in the DNA histogram.
>
Dr. M. Ormerod
We noticed this several years ago when we examined our blood in our
department. Some showed two peaks but some others do not. All of the
examinee were hematologically normal. In our experience, the DIs were less
than 20% apart. We used a detergent method to prepare unfixed nuclei.
Another experience: When leukocytes are fixed in 70% ethanol that contains
RNase, some of the cells move out of the G1 peak to form several peaks of
lower DIs. The process is slow and requires a month or two before peaks are
formed. An important requirement is to use a very dilute PI solution for
staining.
Manabu
******************************
Dr. Manabu Takahashi
Professor emeritus
Yamaguchi University, Ube, Japan
e-mail: manabu32@ymg.urban.or.jp
******************************
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