Chapter 3 78 Requirement Please Include Requested Information 3.3. Instrument configuration and settings The ISx was equipped with three objectives (20x/40x/60x) and 1 CCD camera. All data were acquired using the 60x objective (numerical aperture of 0.9 – pixel area of 0.1 µm2) with fluidics settings set to “low speed/high sensitivity”. We adjusted the default core size of 7 µm to 6 µm using the “Defaults Override” option within INSPIRE software (version 200.1.681.0), as recommended by the manufacturer. Data were acquired over three minutes for standardization among samples with the autofocus setting activated and the “Remove Speedbead” option unchecked. BV421 fluorescence signals were collected in channel 1 (435–505-nm filter), CFSE signals in channel 2 (480–560nm filter) and APC signals in channel 5 (642–745-nm filter). Channel 4 was used as the brightfield channel, and channel 6 (745–780-nm filter) was used for SSC detection. Excitation lasers were set as follows: 405 nm: 120 mW, 488 nm: 200 mW, 642 nm: 150 mW, and 775 nm (SSC): 1.25 mW. Particle enumeration was achieved through the advanced fluidic control of the ISx coupled with continuously running SBs and application of the “objects/mL” feature within the ISx Data Exploration and Analysis Software (IDEAS®). 4.1. List-mode data files IFCM files can be obtained by contacting the corresponding author. 4.2. Compensation description Fluorescent events from singly stained PPP samples were used in the setting of compensation matrices (to compensate for spectral overlap between fluorochromes) such that straight fluorescent populations were obtained when depicted in scatterplots. The following compensation matrix was established for all fluorophores used in this manuscript: 4.3. Data transformation details Arbitrary BV421, CFSE and APC fluorescence intensities were converted to ERF units using 500 nm Rainbow Calibration Particles (RCP-05-5, lot AL01, Spherotech) with known Equivalent number of Reference Fluorophores (ERF) values for C30/FITC/APC. For each detection channel, the MFI of each peak from the four bead populations (1 blanc – 3 fluorescent) were measured, and a linear regression analysis was performed of the log(10) of these values against the log(10) of the known ERF values. The resulting equations were used to convert BV421/ CFSE/APC fluorescent intensities into ERF units.
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