Figure 4 | Scientific Reports

Figure 4

From: Increasing cell culture density during a developmental window prevents fated rod precursors derailment toward hybrid rod-glia cells

Figure 4The alternative text for this image may have been generated using AI.

Single-cell qRT-PCR and immune-cytochemistry analysis of cell culture density impact in rod precursors. The scheme illustrates the generation of 1 × and 4 × cell culture densities from PN0 mice. Horizontal arrows indicate the time of patch-clamp recordings (E-phys) and cell collection for single-cell quantitative RT-PCR (E-phys and scqRT-PCR) from DIV2, DIV4, and DIV8 cultures; (b–e) circles plot − ΔCt values—(CtGene − CtActb); (see “Materials and methods”—“Single-cell real-time qRT-PCR”) for c-Kit (N = 14) (b), Mcam (N = 12) (c), Tnfsf9 (N = 29) (d), Rho (N = 29) (e) and Hcn1 (N = 29) (f) in 29 functionally-characterized single GFP+ rod precursors. (g) Ct values for the housekeeping gene Actb (N = 29). Open and filled circles plot data from cells cultured at lower (1 ×) and higher (4 ×) densities, respectively (see “Materials and methods”). Diamonds plot − ΔCt values for GFP+ cells with functional properties akin to MG (see text and Supplementary Fig. S1). *****P < 0.00001 and ***P < 0.001 for c-Kit and Mcam, respectively. Two-way ANOVA for the effects of DIV and cell density (for cell density, F = 70.55 with 1, 10 df for c-Kit and F = 45.02 with 1, 8 df for Mcam) followed by Bonferroni’s test). *P < 0.05 for the effect of DIV on Rho expression by two-way ANOVA for the effects of DIV and cell density (F = 3.96 with 2 and 22 df), followed by Bonferroni’s test for multiple comparisons indicating a significant difference (t = − 2.74594 with 2, 14 df: P = 0.03538) for ΔCt average difference between PN0/DIV2 and PN0/DIV8 cells (− 5.87 ± 2.15). (h–k) c-Kit immunostaining of DIV8 cells plated at 1 × (h) and 4 × (j) cell culture density along with GFP signal (i,k). Arrowheads point to cells shown at an expanded scale in the inset. Note the lack of c-Kit staining in cells plated at 4 × density compared to the 1 × cells. (l–o) Rho staining of DIV8 cells plated at 1 × (l) and 4 × (n) density along with GFP signal (m,o). Arrowheads point to cells shown at an expanded scale in the inset. Note Rho similar staining in cells plated at 4 × and 1 × cell density.

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