Yen-Chi, Bone marrow derived DCs will always exhibit high levels of autofluorescence, but this is no need for alarm. Many people have the poor habit of insisting all negative populations lie at the X-Y intercept, and that they must be able to analyze all data with quadrant markers. This is a poor habit that must be broken. Once you've worked past this point, data analysis is much simpler. First, do all instrument setup with single stained splenocyte tubes stained with anti-CD8. This will allow you to set your PMT voltages and compensation appropriately. Do not worry that your unstained DCs don't fall where unstained T cells lie, this is unimportant. ***Compensation does not change as the autofluorescence of a population changes as long as you DO NOT adjust the PMT voltages. FITC is FITC no matter what cell it is bound to.**** Next, I would suggest modifying your staining regimen. Bone marrow-derived cultures generate large numbers of dead cells which make analysis virtually impossible. Adding 7-AAD to each tube will allow you to eliminate those dead cells from your analysis regions. As a primary gate, look at a plot of FSC versus 7-AAD. Draw a region around the larger, 7-AAD negative cells. It is important to know that DCs from culture will stain dimly with 7-AAD when alive. I assume this is due to nucleic acids bound to scavenger receptors on the surface of the cell, but have not performed experiments to support that claim. Regardless, don't be alarmed that it appears your DCs have taken up some 7-AAD. Send that gate to a plot of MHC-Class II versus CD11c. Draw a region that encompasses all of the CD11c positive events. Create a logical gate containing only events within both of the previously created regions, and send that to a plot of Class II MHC versus CD86. Immature and mature DCs should now be clearly distinguished based on CD86 and class II expression. Use regions around these populations to generate statistics. To help you decide where to draw these regions, use one-off controls. Include all the antibodies you use in your tube except one (for example, CD86), and gate as I described before. Set your regions appropriately, then run the tube with all the antibodies. You will see shifts in the level of autofluorescence as you treat your cells with maturation stimuli such as LPS, so be sure to run the appropriate controls for your experiment. Best of luck. Kb -- Keith Bahjat, Ph.D. Applications Engineering Manager Cytomation, Inc. Fort Collins, Colorado USA keithb@cytomation.com on 11/15/01 11:44 PM, Yenchi_Chen@BD.com at Yenchi_Chen@BD.com wrote: > 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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