Fig 1: Protein expression in tear film before and after Femto-LASIK. (A) Evaluation of MMP-9 values pre-OP as well as 5 and 90 days after Femto-LASIK revealed no significant differences. (B) The NGF level at all three investigation points was nearly the same. (C) The amount of the pro-inflammatory cytokine IL1-β was not altered following Femto-LASIK. (D) The same levels for the pro-inflammatory chemokine IL-8 were identified in all samples. (E) The CGRP level at 5 days post-OP was significantly downregulated in comparison to pre-OP, but not in comparison to 90 days post-OP. Values are mean ± SEM ± SD; * p < 0.05. Abbreviations: CGRP = calcitonin gene-related peptide; IL = interleukin; MMP-9 = matrix metalloproteinase-9; NGF = nerve growth factor.
Fig 2: (A,B) Nestin and NeuN immunofluorescence staining showed changes in cell morphology on days 4 and 7; (C) Neurites’ number, mean length, and total length analysis, n = 8; (D) ELISA results for the expression of BDNF and NGF in pg/mL. Note (*: p < 0.05/**: p < 0.001).
Fig 3: Neurotrophic factors expressed by hPMSCs. Western blotting was performed to analyze the synthesis of pro-BDNF (32 KDa), pro-NT3 (35 KDa), pro-NGF (32 KDa) in hPMSCs cultures at passage 3 (P3), passage 4 (P4), passage 5 (P5), grown in normoxia (N) and hypoxia (H). hPMSCs were cultured in (A) DMEM high glucose (4,5 gr/L) or (B) NB-B27 media. Expression of the proteins was quantified as arbitrary units using ImageJ software. Values are expressed as the ratio between the corresponding pro-factor and β-actin as a loading control (on the right). Two-way ANOVA and post hoc Sidak’s test statistical analysis was performed (****p ≤ 0.0001; *p ≤ 0.05). Maintenance of hypoxic conditions was monitored by immunodetection of hypoxia inducible factor (HIF). Secreted BDNF (C) and NGF (D) were measured using ELISA-based assays. ANOVA and post hoc Dunnet’s test. *p < 0.05, **p < 0.005, ***p < 0.001 and ****p < 0.0001.
Fig 4: Detection of IL-6 and NGF using EliChip™. Side-by side validation of EliChip™ compared to ELISA through detection of IL-6 from A. M0 macrophages B. M1 macrophages. C. Quantification of IL-6 produced by chondrocytes of healthy cartilage constructs (HC-S) and OA-like model at day 14 and day 28 using EliChip™. D. Quantification of NGF expressed by chondrocytes of HC-S and OA-like model at day 14 and day 28 using EliChip™. The statistical analysis was performed using Shapiro-Wilk test followed by Sidak's Multiple comparison test. The statistical significance level was set to p < 0.05; ns = non-significant. For each assay n = 3; 3 different donors.
Fig 5: TWEAK induces proliferation, migration and cytokine production in HFSCs. Human HFSCs were cultured in vitro and stimulated with TWEAK (0–250 ng/mL). (A) Proliferation of HFSCs analysed via flow cytometry. n = 5 per group. (B) Cell migration assessed via scratch analysis. n = 5 per group. (C) mRNA expression levels of EGF, BFGF, TGF‐β, NGF and VEGF measured via qRT‐PCR. n = 3 per group. (D) Cytokine levels in the supernatants determined via ELISA. n = 3 per group. Data represent mean ± SEM from three to five independent experiments. *p < 0.05 compared with the 0 ng/mL group. #p < 0.05 compared with the 10 ng/mL group. HFSC, hair follicle stem cells; qRT‐PCR, quantitative real‐time PCR; SEM, standard error of the mean; TWEAK, tumour necrosis factor‐like weak inducer of apoptosis.
Supplier Page from Abcam for Human beta Nerve Growth Factor ELISA Kit