Author: 1Willem E. Corver and 2Jan van der Aa Affiliation: 1 Department of Pathology Leiden University Medical Center Leiden, The Netherlands 2 Becton Dickinson European HQ Immunocytometry Systems Erembodegem, Belgium Correspondence: Willem E. Corver, PhD Leiden University Medical Centre Department of Pathology The Netherlands E-mail: W.E.Corver@lumc.nl

Adapted from (with permission of the publisher): Cytometry: 39:96-107 (2000)

A standard two-laser FACSCalibur does not allow simultaneous FL3-A/W pulse-processing and FL4-H fluorescence detection. For this purpose, the FACSCalibur needs to be modified. This modification can be useful for the simultaneous measuring FITC, RPE and APC fluorescence in combination with PI as DNA stain.

Open the hood of the FACSCalibur and disconnect the following wires from the main boards according to the following instructions:

1. 1. Disconnect "Comp FL3 in J-200" of main board J4,
2. 2. Disconnect "ADC Width" of main board J5,
3. 3. Disconnect "Width" of main board J10,
4. 4. Disconnect "FL4-H" of main board J10
5. 5. Connect it to "Comp FL3 in J-200" of main board J4 (5),
6. 6. Connect the "Width" of main board J10 with a new electrical wire to the "ADC Width" of main board J5 (6).

Switch of the red diode laser in order to change the FL3-W gain (P7). FL3-W instruments settings will be maintained if the red diode laser is turned on again, but P7 (FL3-W) is inactive (gray) in the "Detectors / Amps" window. Note that the detectors of P5 and P7 have been interchanged (P5 = FL4 and P7 = FL3). The modes of P5 and P7, in the "Detectors / Amps" window, must be "Log" and "Lin" and the P5 and P7 parameter labels must be changed into FL4-H and FL3-W, respectively.