• Mon. May 23rd, 2022

MMP-1 cycle thresholds were quantified by comparison to an MMP-1 standard curve generated using known MMP-1 concentrations and standardized to 18S rRNA


Mar 21, 2022

MMP-1 cycle thresholds were quantified by comparison to an MMP-1 standard curve generated using known MMP-1 concentrations and standardized to 18S rRNA. epithelial cell migration in a wound-healing assay, and this too was matrix metalloproteinase-dependent, since it was blocked by the matrix metalloproteinase inhibitor GM6001. In summary, we report a novel mechanism by which 21-mediated signals from the ECM modulate matrix metalloproteinase-1 secretion by HBECs, regulating their migration and epithelial repair in TB. and standards and cell supernatants were incubated with DQ type I collagen for 24?h. Gain of fluorescence originated from DQ collagen degradation. Mean basal collagenase activity from controls was subtracted from CoMtb-stimulated wells without ECM and type I collagen, respectively, and fold-change in collagenase activity was calculated relative to CoMtb-stimulated wells in the absence of ECM. MCH-1 antagonist 1 Reverse Transcription and Real-Time PCR After 6?h incubation, HBEC cells were lysed with TRI-reagent and total RNA extracted using PureLink RNA Mini Kit (Thermo Fisher Scientific). 1?g RNA was reverse transcribed using QuantiTect Reverse Transcriptase Kit (Qiagen, Manchester, UK) and qPCR reactions were performed in the ABI Prism 7700 (Applied Biosystems, Paisley, UK). MMP-1 cycle thresholds were quantified by comparison to an MMP-1 standard curve generated using known MMP-1 concentrations and standardized to 18S rRNA. MMP-1 primers and probes were custom made and supplied by Sigma-Aldrich (forward primer: 5-AAGATGAAAGGTGGACCAACAATT-3; reverse primer: 5-CCAAGAGAATGGCCGAGTTC-3; probe: 5-FAM-CAGAGAGTACAACTTACATCGTGTTGCGGCTC-TAMRA-3) and 18S rRNA primer and probe mix was supplied by Thermo Fisher Scientific. Flow Cytometry HBEC cells were detached with 5?mM EDTA, washed with PBS, fixed with 4% paraformaldehyde (w/v), and blocked with 1% BSA/5% human serum (v/v) buffer. Cells were incubated for 1?h at room temperature with FITC-conjugated anti-human integrin antibody or mouse IgG1 isotype control antibody. Flow FGF2 cytometry was performed on a FACSCalibur (BD Biosciences, UK) cytometer. The baseline Forward Scatter, Side Scatter, and FL1H settings were adjusted using an unstained, unstimulated cell sample. Mean fluorescence intensities (MFI) were compared after normalization to the isotype control. Data were analyzed using FlowJo vX.0.6 (Tree star, Ashland, OR, USA). Confocal Microscopy 4-well glass slides were pre-coated with Coll-I and HBEC cells seeded and incubated as described. Cells were fixed, blocked, and stained with FITC-conjugated anti-integrin or mouse IgG1 isotype control antibody. MMP-1 was stained with a rabbit anti-human MMP-1 primary antibody (Millipore) and Cy5-goat anti-rabbit IgG (Abcam) as secondary antibody. Staining with secondary antibody alone was used as control. For F-actin staining, cells were permeabilized with 0.5% saponin (v/v) and stained with phalloidin conjugated with Alexa Fluor 594 (Thermo Fisher Scientific). DAPI was used as nuclear counterstain. Slides were scanned using a 63 oil immersion objective and to avoid bleed-through effects, each dye was scanned independently in a Leica TCS SP5 confocal microscope equipped with 405?nm diode laser, 488?nm argon laser, 543 and 633?nm HeNe lasers, and using the Leica Application Suite 2.6.2 software (Milton Keynes, UK). Images were edited using ImageJ software v1.46r (NIH, MD, USA). Wound Healing Assay 96-well ImageLock plates (Essen Biosciences Ltd., Hertfordshire, UK) were coated with Coll-I or poly-l-lysine and cells were seeded as described. A standard wound was created in each cell monolayer using a WoundMaker (Essen Bioscience Ltd.) according to manufacturers MCH-1 antagonist 1 protocol. Cells were stimulated and/or soluble Coll-I was added (sColl-I). Plates were incubated in the IncuCyte Zoom (Essen Biosciences Ltd.). Images were taken at 2?h intervals for a maximum of 24?h. Histology and Immunohistochemistry Lung MCH-1 antagonist 1 biopsy specimens were collected as part of routine clinical care of patients who were subsequently diagnosed with TB. Patients gave consent for the residual paraffin embedded tissue blocks not required for diagnostic purposes, to be analyzed in this study. The study was approved by the NHS National Research Ethics Service (13/LO/0900) and University College London Hospitals Joint Research Office. Human control or Mtb-infected lung tissue blocks were cut into 5?m sections and stained with Hematoxylin and Eosin (H&E) or modified MCH-1 antagonist 1 Trichrome using an automated slide-stainer (Sakura, Tissue-Tek DRS 2000). In brief, sections were stained.