Hi Yenchi, That's pretty much normal, the background "signal" from non-autofluorescent cells is essentially noise, so the fl1/fl2 plot for say lymphocytes would be a plot of two noise patterns, and it would be uncorrelated, forming a roundish pattern. In the case where there is a true fluorescent signal (even if it is an unwanted one) that can be read in both detectors (as is the case with cellular autofluorescence), then the pattern would be a correlated diagonal such as the one that you have. Depending on how strong the signal is relative to noise, the diagonal would vary in width along its axis. The angle of the diagonal depends on the colour of the fluorescence. Ray > From: Yenchi_Chen@BD.com > Date: Fri, 16 Nov 2001 12:44:38 +0800 > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Subject: Dendritic cell analysis by FCM > > I would like to ask any of you to provide expertise for my clients on > cultured bone marrow derived dendritic cell analysis by flow cytometers. We > found the background was very strong using unstained cells. Therefore, we > had to lower the voltage of PMT(FL1, FL2) to approximately 420-250. > However, the negative area of dot plot FL1 vs FL2 showed narrow elliptical > shape toward diagonally. It did not show the round or oval shape as many > other white blood cell clusters did.The antibodies were used - > CD11c-PE(data.oo2) and CD86-FITC(data.003). Data.001 is unstained sample > for negative control. The staining buffer is PBS with 1%FBS and 0.05% NaN3. > Please advise. > Thank you very much. > Yen-Chi Chen > BD Taiwan, BDB-COE > Tel: 02-2722-5660 ext 297 > Fax: 02-2725-1768 > yenchi_chen@bd.com > (See attached file: Data.003)(See attached file: Data.002)(See attached > file: Data.001)
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