Hi Katja, this is almost certainly due to bad alignement of the red laser in the FACSCalibur. You have to make sure that the dichroics are well aligned and then adjust the timing between the two lasers very delicately (adjust the red laser height adjustment). In general doing this with the Calibrite Beads does often not result in correct alignment because the PerCP beads do not exhibit the same compensation characteristics as do tandem conjugates. It is therefore better to adjust your laser while running real 4 color samples through the machine, and adjust the laser + dicroics until you get optimal compensation (going back and forth between the cells and APC beads to adjust the time delay after you touch the laser). You should never have to go much over 50% compensation of FL4-FL3 with PE-CY5 conjugates (even with bad conjugates). Of cours there is a variability in the batches of tandem conjugates due to different conjugation efficiencies, but in most cases I have seen the problems are due to bad timing adjustments. If you do not know how to do this yourself (I agree it's a bit tricky and time consuming), ask the technician to get the job done until your cells look good. Don't let them get away with just showing you on beads that the things work..;) Hope that helps. Matthias _____________________________________ Prof. Dr. Matthias Haury Instituto Gulbenkian de Ciencia Unidade de Imagiologia Celular Rua Da Quinta Grande N° 6 - Apt.14 P-2781 Oeiras Codex Portugal Email: mhaury@igc.gulbenkian.pt Tel: + 351 21 440 79 85 Office Tel: + 351 21 440 79 10 Secretary Fax: + 351 21 440 79 70 _____________________________________ At 13:35 +0100 26/11/1999, Katja Adolf wrote: > Dear flowers, We are currently using a 4 colour panel with FITC, >PE, RPE-Cy5 and APC on a FACS Calibur and have to compensate as much as >86% FL4-FL3 to get reasonable populations. This worked until a new >antibody batch and combination differed too much in flourescense. Also we >are not happy with this high compensation. Markers of interest are FITC >and PE labeled, but we would like to use the FL3 and FL4 for more than >just gating purposes. Looking in the archive there is only an unanswered >post from 1995, claiming that it probably is impossible NOT to use PerCP >but tandem conjugates instead. Any others using this setup? Any >advices? Thanks in advance. With best regards, Katja >************************************************** >Katja Adolf, PhD student >Aarhus University Hospital >Allergy and Lung Research >Clinic >Nørrebrogade 44, DK-8000 Aarhus C, Denmark >Phone +45/ 8949 2106 Fax +45/ 8949 2110 >email: kad@mailme.dk >**************************************************
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