Thanks so much for the response to the problem I've posted earlier.
There were still a lot of questions in the answers I've received, so I
thought I'd be more specific on the protocol used:
Cells were derived from whole lung collagenase/DNase/EDTA digests
1. all cells were pre-treated with mouse FcR-block (24G2), followed by
staining with anti-CD11c-PECy5 + anti-MHCII-PE or isotype-PE
2. debris was gated out using FSC/SSC pattern and lung cells were gated
for CD11c-positivity
3. within CD11c-high cells, autofluorescence was assessed in the free
FL1 channel: a low and a high autofluorescent peak could be
distinguished (this is also the case in cells unstained for a -PE
marker)
4. MHCII expression vs isotype was checked in each of these 2 fractions.
The intensities are shown in the table below:
isotype-RPE anti-MHCII-RPE
low autofluorescence
CD11c+ cells 1st decade 2nd-3rd decade
high autofluorescence
CD11c+ cells 2nd decade 2nd decade
The question was originally: are the high autofluorescent cells positive
for MHCII (2nd decade), with the signal "drowned" in high
autofluorescence?
Or should any real MHCII-positivity be emerging on top of background
autofluorescence?
-> The consensus idea is that the high autofluorescent cells are
negative for MHCII and any real positivity should indeed be added on top
of autofluorescence. Any further comments are very welcome.
Thanks to Mario Roederer, Dirk Van Bockstaele, Olindo Onassis, Simon
Monard and Leonid Volkov.
Karim Vermaelen
Pulmonary Immunobiology
Ghent University Hospital
Belgium
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:56:11 EST