enzchek gelatinase collagenase assay kit  (Fisher Scientific)

Journal: Bioactive Materials

Article Title: Glycosaminoglycan-functionalized hydrogels for sustained delivery of tissue inhibitor of metalloproteinase-3 mediating matrix metalloprotease inhibition and extracellular matrix stabilization

doi: 10.1016/j.bioactmat.2026.02.010

Figure Lengend Snippet: TIMP-3 from GelMA/sHAc hydrogels reduces matrix degradation ex-vivo. (A) Schematic overview of the ex-vivo matrix degradation assay. Human dentin slices were demineralized to expose the collagen matrix and activate endogenous proteases and subsequently incubated with TIMP-3-containing or TIMP-3-free hydrogels to assess their effects on native matrix degradation. (B) The potential of TIMP-3 released from hydrogels after 24 and 168 h to influence native matrix turnover was analyzed in an ex-vivo dentin slice model using the EnzChek assay, in comparison to hydrogels without TIMP-3. Demineralized dentin slices without additional treatment served as controls (Ctrl). (C) Collagen degradation by endogenous matrix-located proteases was assessed in the presence or absence of hydrogel released TIMP-3 or hydrogels without TIMP-3. Collagen preservation was measured by Sirius red staining followed by dye elution. Inactivated dentin slices from autoclaved teeth served as reference. One-way ANOVA: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001., For (A) a = ∗∗∗p < 0.001 vs. Ctrl, for (B) b = ∗∗∗p < 0.001 vs. Reference. Significant differences were determined versus Ctrl/Reference and between hydrogels with or without TIMP-3 of the same composition to distinguish material-from TIMP-3-specific effects. (D, E) TIMP-3-loaded GelMA/sHA c hydrogels reduce ECM degradation in a human ex-vivo skin model. (D) Schematic of the ex-vivo human skin model. 10 mm skin biopsies with a 4 mm wound were collected within 24 h post-mortem and cultured ex-vivo. Pathological ECM degradation was induced by collagenase and TNF-α treatment. GelMA/sHA c hydrogels with or without TIMP-3 were applied to the wound surface and cultured for 72 h. (E) Overview of sample groups and representative Sirius red-stained histological sections showing ECM organization: intact wound (no pathological ECM degradation), wound with induced ECM degradation treated with GelMA/sHA c , or GelMA/sHA c + TIMP-3. The wound region is indicated by a dotted line. (F) Remaining collagen content quantified by Sirius red staining of human skin samples, followed by dye elution and absorbance measurement. Absorbance values were corrected for initial sample weight differences. One-way ANOVA: ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: To stimulate protease secretion, cells were treated with 20 ng/mL human recombinant TNF-α (Bio-Techne, Wiesbaden, Germany) for 48 h. The supernatants were collected and used to determine the protease activity in the presence or absence of soluble HA c and sHA c with or without additional TIMP-3 or hydrogel extracts as described in section “cytotoxicity of hydrogel extracts” using the EnzChek Gelatinase/Collagenase Assay Kit (Fisher Scientific, Schwerte, Germany) according to the manufacturer's protocol.

Techniques: Ex Vivo, Degradation Assay, Incubation, Comparison, Preserving, Staining, Cell Culture

Journal: Bioactive Materials

Article Title: Glycosaminoglycan-functionalized hydrogels for sustained delivery of tissue inhibitor of metalloproteinase-3 mediating matrix metalloprotease inhibition and extracellular matrix stabilization

doi: 10.1016/j.bioactmat.2026.02.010

Figure Lengend Snippet: TIMP-3 maintains protease inhibitory activity in the presence of sHA c and hydrogels release bioactive TIMP-3. (A-D) Influence of soluble GAGs and hydrogel extracts on TIMP-3-mediated inhibition of protease activity in TNF-α-stimulated NHDFs. (A) Schematic of the experimental design. Inflammation was modeled by stimulating NHDFs with TNF-α, inducing increased protease secretion. Gelatinase/collagenase activity in supernatants was quantified using the EnzChek assay with a fluorogenic gelatin substrate in the presence or absence of soluble TIMP-3, soluble GAGs or hydrogel extracts. (B) Protease activity in the supernatants after TNF-α treatment relative to unstimulated controls. (C) Protease activity of TNF-α-stimulated supernatants incubated with soluble GAGs (HA c , sHA c ) with or without TIMP-3. (D) Protease activity of TNF-α-stimulated supernatants incubated with hydrogel extracts (prepared by 72 h hydrogel incubation in medium) in the absence or presence of TIMP-3. One-way ANOVA: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Only significant differences relative to the Ctrl without TIMP-3 or relative to TIMP-3 alone are shown in C/D. (E) The inhibitory potential of TIMP-3 released from the hydrogels was measured using a MMP-9 activity assay. (F) The ratio of bioactive TIMP-3 to the total amount of released TIMP-3 was calculated and expressed as a fold change relative to GelMA hydrogels without GAGs. (G) Collagen-based ECMs were incubated with collagenase (CHC) for 20 or 60 min with TIMP-3 released from the hydrogels after 24 or 168 h. The remaining collagen was detected after Sirius red staining and elution. Two-way ANOVA for A, B: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. One-way ANOVA for C: ∗p < 0.05. (H) Molecular rationale for the regulatory role of sHA c on TIMP-3-mediated protease inhibition. The MD-refined complex of TIMP-3 (in grey) with HA6_3AC1 (atom-colored brown sticks, color gradient as in D) is shown superimposed with the TIMP-3/ADAM complex (PDB ID 3CKI ). ADAM is shown in green, and the corresponding TIMP-3 structure has been omitted for clarity.

Article Snippet: To stimulate protease secretion, cells were treated with 20 ng/mL human recombinant TNF-α (Bio-Techne, Wiesbaden, Germany) for 48 h. The supernatants were collected and used to determine the protease activity in the presence or absence of soluble HA c and sHA c with or without additional TIMP-3 or hydrogel extracts as described in section “cytotoxicity of hydrogel extracts” using the EnzChek Gelatinase/Collagenase Assay Kit (Fisher Scientific, Schwerte, Germany) according to the manufacturer's protocol.

Techniques: Activity Assay, Inhibition, Incubation, Staining

Journal: STAR Protocols

Article Title: Protocol for isolating viable human central nervous system T cells

doi: 10.1016/j.xpro.2026.104464

Figure Lengend Snippet: Temperature-induced gene artefacts Bulk RNA sequencing was performed on matched subcortical white matter (WM) CD11b + microglia and CD69 + CD8 + T cells from n = 4 brain donors. (A) Experimental overview. (B) Principal component analysis (PCA) of microglia and T cells using different dissociation methods. (C) Barcode plot showing enrichment of temperature induced signatures in microglia and T cells following collagenase-IV treatment at 37 0 C without transcription and translation inhibitors. (D) Gene expression of major brain T RM -cell markers after different dissociation procedures. (E) Expression of genes associated with T cells ( CD3D , CD8A ), B cells ( CD19 ), microglia ( P2RY12 ), and neurons ( MAP2 ) in the sequenced samples, obtained by RNA sequencing. Genes associated with astrocytes ( AQP4 ) and oligodendrocytes ( MAG ) were not detected. (F) Log-fold change of WM compared to circulating effector memory T (EM) cells (from Hsiao et al. ) to the heat-induced signature of T cells. Col-IV, collagenase IV; CPM, counts per million; FC, fold change; Inh., transcription and translation inhibitors; MG, microglia; T, T cell; T RM , tissue-resident memory T cell; WM, white matter.

Article Snippet: Collagenase IV , Worthington , Cat# LS004189.

Techniques: RNA Sequencing, Gene Expression, Expressing