Chapter 3 70 Framework Criteria What to report Please complete each criterion 3.4 Isotype controls. The use of isotype controls is applicable to immunofluorescence labelling only. State whether isotype controls were analyzed at the same settings and during the same experiment as stained samples. If utilized, state which antibody they are matched to, the concentration used, and what the results were (Section 4.2, 4.3, 4.4). Due to conjugation differences between manufacturers if should be stated if the isotype controls are from the same manufacturer as the matched antibodies. Isotype controls samples were measured at the same dilution and at the same concentration as matched stained controls and were recorded during the same experiment at the same imaging flow cytometer with acquisition settings similar to all other samples, including laser power and flow rate. No substantial changes in fluorescence signal were observed between unstained and matched isotype controls. Isotype - mAb matching: IgG1,k-BV421, clone MOPC-21 (100 µg/mL, BioLegend) matched with anti-human CD31–BV421, clone WM-59 (50 µg/mL, BioLegend); IgG1,k-APC, clone MOPC-21 (200 µg/mL, BioLegend) matched with anti-CD9–APC, clone HI9a (6 µg/mL, BioLegend, San Diego, USA); anti-CD63–APC, clone H5C6 (200 µg/mL, BioLegend); and antiCD81–APC, clone 5A6 (200 µg/mL, BioLegend), IgG2a,k-APC, clone RTK2758 (200 µg/mL, BioLegend) matched with anti-mouse CD31-APC, clone 390 (200 µg/mL, BioLegend). No isotype control for CFDA-SE was used. All isotype controls are from the same manufacturer as the matched antibodies. 3.5 Single-stained controls. State whether single-stained controls were included. If used state whether the singlestained controls were recorded using the same settings, dilutions, and during the same experiment as stained samples and state what the results were, preferably in standard units (Section 4.2, 4.3, 4.4). Single-stained control samples were included for every mAb used in this work, and were measured at the same dilution and at the same concentration as matched stained controls and were recorded during the same experiment at the same imaging flow cytometer with acquisition settings similar to all other samples, including laser power and flow rate. Single-stained controls aided in the establishment of the compensation matrix (to eliminate spectral overlap between detection channels). The following results were obtained for a representative single-stained PPP sample: anti-CD9, anti-CD63, anti-CD81 (mix) Counts: 7666, Median Fluorescent Intenstiy: 906, Equivalent number of Reference Fluorophores: 52 - CFSE Counts: 3234, Median Fluorescent Intenstiy: 816, Equivalent number of Reference Fluorophores: 134 - anti-human anti-CD31 Counts: 3341 Median Fluorescent Intenstiy: 4101, Equivalent number of Reference Fluorophores: 12701.
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