Quantitative Assessment of Fluorescent Reporter Expression in 3D Retinal Organoids

Human iPSC-derived retinal organoids develop robust Alzheimer’s disease neuropathology

“…Drug development and biomarker validation applications necessitate both a robust model and simple, sensitive, and quantitative assays for outcome measures. To provide proof of principle of the ability to perform quantitative analyses of amyloid in whole 3D ROs, we made use of a technology we previously developed and validated termed 3D Automated Reported Quantification (3D-ARQ) ( Vergara et al, 2017 ; Vielle et al, 2023 ). This technology is based on the quantification of total fluorescence intensity with high speed and high sensitivity.…”

“…This technology is based on the quantification of total fluorescence intensity with high speed and high sensitivity. We had also identified RO size as the major source of variability in fluorescence intensity outputs in ROs and devised a method for size normalization using a globally expressed fluorophore ( Vergara et al, 2017 ; Vielle et al, 2023 ). Therefore, we performed whole mount staining of CTR-ROs (A18945) and AD-ROs (derived from the HVRDi001-A-1 line) after 3 months of differentiation with NIAD-4 to stain amyloid, and we determined the best parameters for 3D-ARQ quantification of total fluorescence intensity using a TECAN Spark plate reader as described in the Methods.…”

“…Retinal organoids at 3 months of differentiation ( n = 6 per condition), were fixed for 1 h in 4% paraformaldehyde and washed three times in PBS. Organoids were then plated in a 96-well, black V-bottom plate at one organoid per well in 1X PBS, and the auto-fluorescent background was measured by 3D-automated reporter quantification ( Vergara et al, 2017 ; Vielle et al, 2023 ). Briefly, a Tecan Spark Plate reader (application SparkControl v2.3) was used, and after determining the Z-positions, fluorescence intensity was measured using the following optimized parameters: Mode: Fluorescence Top Reading; Excitation wavelength: 475 nm; Excitation bandwidth: 5 nm; Emission wavelength: 625 nm; Emission bandwidth: 5 nm; Gain: 100, Manual; Number of flashes: 20; Integration time: 40 μs; Lag time: 0 μs (Fluorobrite DMEM filled wells were used as blank).…”

“…The plate fluorescence intensity was reassessed using the same parameters. For size normalization ( Vergara et al, 2017 ; Vielle et al, 2023 ), nuclear staining with 10 μg/ml Hoechst 33342 (Invitrogen, H3570) was performed for 1 h at room temperature, followed by three 15 min washes in 1X PBS. After three 15 min washes, the organoids were incubated overnight in 80% glycerol at 4°C, washed 3 × 15 min in PBS, washed a final time in DMEM Fluorobrite, and fluorescence intensity was assessed using excitation wavelength: 350 nm; Excitation bandwidth: 5 nm; Emission wavelength: 461 nm; Emission bandwidth: 5 nm.…”

“…All cryosections were counterstained with DAPI (b,e,h,k). ( m Vielle et al, 2023). Therefore, we performed whole mount staining of CTR-ROs (A18945) and AD-ROs (derived from the HVRDi001-A-1 line) after 3 months of differentiation with NIAD-4 to stain amyloid, and we determined the best parameters for 3D-ARQ quantification of total fluorescence intensity using a TECAN Spark plate reader as described in the Methods.…”