We use cyclosporin routinely without much problem (assuming you're
starting with cells from a normal individual). Transformation
efficiency is usually high. I never heard of using OKT3; I would guess
that the early morphologic changes that you see are the result of T cell
activation induced by the antibody; the method may actually work by
inducing anergy in any potentially EBV reactive T cells present. The
other method that works very well is to deplete your culture of T cells
prior to transformation.
Fred Garbrecht
Medical College of Wisconsin
fgarbrec at post.its.mcw.edu
On 5 Jan 1993, Cheung C. Yue wrote:
> Date: 5 Jan 93 23:51:15 GMT
> From: Cheung C. Yue <ccy at po.CWRU.Edu>
> To: immunology at net.bio.net> Subject: Lymphocyte transformation by EBV - help wanted
>>> It has been a while since I did EBV transformation. Previously I had
> used OKT3 (?to shut off autologous CTL response), but the last several
> times I did this (in '89!!), I used cyclosporin as per a number of
> people's suggestions. Well, the last 3 times I used cyclosporin I got
> no transformed cell lines, but using OKT3, I had at least 1 out of 4
> tries. When I look at the cells, the OKT3 treated cultures developed
> clustering of cells by the second day, and the media gets quite acidic
> within a week. In contrast there is not much cell clustering in the
> cyclsporin treated cells even after 1 week. Interestingly the cyclosporin
> treated cells have on several occassions developed a bunch of large
> granular-appearing cells which tended to stream together forming a
> streak of cells (looks like a cotton fiber when out of focus, but is
> actually a bunch of cells that line up in a tubular kind of structure).
> Are these macrophages? Have others observed this phenomenon? Are the
> clusters of cells in the OKT3-treated samples early transformed cells?
> I kind of doubt the last point since the clusters do not have the same
> appearance of the clusters of LCL, and they appear way too soon.
>> Anyone care to comment? I would welcome getting in touch with anybody
> on the net with experience transforming lymphocytes. As it turns out,
> nobody in Cleveland is actually doing lymphocyte transformation at present,
> so I can't bug anybody local about this. Thanks for all info.
>> C Cho Yue
>ccy at po.cwru.edu> --
>
