Table 3.

Troubleshooting.

Steps	Problem	Possible reason	Solution
5	Cells do not proliferate and begin to differentiate.	Activity of EGF and/or bFGF is lost.	Avoid multiple freezing/thawing cycle for EGF/bFGF stocks.
10	The transfection rate is low.	Low titer of lentiviral vector, poor cell condition, negative impact on cell proliferation of APPSL or PSEN1(ΔE9).	Increase titer of lentiviral vector. Do multiple transfections with the same lentiviral vector.
18	FACS-sorted cells are not proliferating or apoptotic.	Expression of FAD mutations obtained with the FACS sorting is too high.	1) Plate the FACS-sorted cells in small-scale dishes to increase the cell density (the high cell density helps to accelerate ReN cell proliferation); 2) Enrich cells with lower fluorescence signal intensities.
34 (A) vi	The color of the ReN differentiation medium rapidly changes into yellow in 1–2 days.	The cell density is too high.	1) Change the 3D cell culture medium more frequently in early period (every 2 days); After 2–3 weeks, switch to a normal medium change schedule (every 3–4 days); 2) Reduce the number of cells at step 33.
34 (B) i	ReN cells sink down to the bottom and look like 2D culture.	Matrigel concentration is too low (since each lot of Matrigel has a different protein concentration, the optimal dilution rate should be determined every time).	1) Use different dilution rate to increase the final Matrigel concentration (Even if the cell body seems to be at the bottom, we observed that the Matrigel still formed 3D gels on top of the cells, making 3D neurite outgrowth and displaying Aβ and tau pathologies. Actually, low Matrigel concentration is desirable for immunohistochemical staining to reduce background). Pre-test 1:10, 1:15 and 1:20 dilutions and pick the best dilution rate for each lot; 2) Prewarm the plates and move them to 37 °C immediately after plating. This will accelerate the gelation of Matrigel before the cells sink down.
36	Aβ aggregates/p-tau pathologies are delayed or not evident.	Number of cells is low.	The starting number of cells/ml is between 3–10 million/ml, but number of cells desired is 10 million/ml.
		Aβ42 levels (Aβ42/40 ratio) are not high.	1) Enrich cells further with higher fluorescence signal intensities; 2) Start 3D culture again using high quality culture of cells with earlier passage number or start again from the viral transfection; 3) We also found that additional transfection with PSEN1(ΔE9) vector could further elevate Aβ levels.
36 (D) (E)	Strong backgrounds	Non-specific interaction between the primary/secondary antibodies and the fixed Matrigel matrix. Uneven solidification of the Matrigel in thin-layer 3D cultures.	Increase permeabilization, blocking and washing time. Decrease secondary antibody concentrations. Use lower Matrigel concentrations (1:15 or 20) during thin-layer 3D culture generation.
36 (F)	Strong backgrounds (Amylo-Glo)	1) The concentration of Amylo-Glo working solution is too high. 2) Presence of autofluorescent aggregates.	Reduce the Amylo-Glo working solution concentration and incubation time. Check the fluorescence background in other flouropore filters including FITC/Rhodamine.
36 (H) (J)	Normalization of total protein levels for Western blot does not fit to control protein levels including GAPDH.	The total protein extracts are contaminated by the solubllized Matrigel components liberated during the extraction.	Use the human specific protein markers such as human-specific mitochondrial antigen (H-mito, see Table 2) to renormalize the samples. Use dot blot analysis.
36 (L)	Poor fibrils imaging	Tau fibril structures are damaged during the extraction. Tau fibrils are not mature.	Reduce the Sarkosyl extraction time at room temperature. Use 3D culture samples differentiated more than 14 weeks.
	Contamination with other non-specific structures.	Sarkosyl extraction was not enough.	Increase the Sarkosyl extraction time. Re- centrifuge the samples before Sarkosyl extraction.