Journal: PLoS ONE

Article Title: Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

doi: 10.1371/journal.pone.0047316

Figure Lengend Snippet: (A–B) Fluorescent histograms of unsorted (total) cells exposed to acute and chronic NGF doses up to 24 hrs, showing an increased % of AnnexinV positive cells (isotype-matched control FI = 7.65). (C) AnnexinV specific Mean Fluorescent Intensity (MFI) for both acute and chronic, as a function of NGF treatments (% over control untreated myoFB, p<.05). (D) Representative pictures depicting monolayers exposed to acute or chronic 100 ng/mL NGF showing perinuclear/nuclear AnnexinV positivity alone (arrow-heads) or in combination with Propidium Iodide (PI; arrows) in stained cells. (E–F) Cytograms showing AnnexinV/PI plotter in cells exposed to acute and chronic exposure to NGF. Untreated myoFB were mainly AnnexinV/PI double-negative (LL, Lower Left quadrant), with a % range between 73 and 85; both acute and chronic NGF treatments resulted in increases of both AnnexinV pos /PI neg (LR, Lower Right quadrant), indicating a proportion of cells undergoing apoptosis. Particularly, upon chronic NGF treatment, a population of cells also progressed to a later stage of apoptosis (AnnexinV pos /PI pos ; UR, Upper Right quadrant).

Article Snippet: To detect externalized PS, cells were probed with FC-AnnexinV-Apoptosis detection kit (Miltenyi), according to the manufacturer’s directions with the exception that cells were post treated with RNAse A and finally fixed in 3% PFA , .

Techniques: Staining

Journal: Scientific Reports

Article Title: A novel technique to determine the cell type specific response within an in vitro co-culture model via multi-colour flow cytometry

doi: 10.1038/s41598-017-00369-4

Figure Lengend Snippet: Investigation of the ability for the herein presented method to obtain a cell suspension of the 3D in vitro triple cell co-culture model to incite cell death, as determined with the Annexin V assay. Following detachment from a micro-porous membrane insert via EDTA-Trypsin treatment the level of cell death ( i.e. either apoptosis or cell death ( e.g. necrosis)) was determined for ( A ) the total cell suspension via two-colour flow cytometry (FACS), or ( B ) for each specific cell type of the co-culture system via five-colour FACS. In both ( A and B ), data shows the viability of the co-culture following 24 hours exposure at 37 °C, 5% CO 2 to either complete medium (negative control), Camptothecin at [0.002 mg/mL] (positive apoptosis control) or extreme freezing (−80 °C for 30 minutes) (positive dead cell ( i.e. necrosis) control). After implementing the specific gating strategy for the co-culture suspension (SI Fig. ) ( C ) indicates the gating strategy used to deduce the viability status of the cell cultures was defined as healthy, early apoptotic, late apoptotic or dead. All data was analysed using FlowJo (Version 10, TreeStar, USA). In ( A and B ) all data expressed is the mean ± standard error of the mean (SEM) of the %Frequency. Experimentation was repeated on three separate occasions in triplicate (n = 3).

Article Snippet: To achieve this, samples were prepared as stated above, however prior to using the Annexin-V-Fluos kit cell suspension samples were firstly incubated with Fc-block receptor (Miltenyi Biotech (Cat #:130-059901), Switzerland) for 10 minutes in order to inhibit Fc-receptor binding of specific antibodies, then washed x1 with PBS prior to being stained for the specific cell surface proteins CD14 (MDM), Pan-Cytokeratin (A549 epithelial cells) and CD1c (MDDC) and incubated at 4 °C for an additional 30 minutes.

Techniques: Suspension, In Vitro, Co-Culture Assay, Annexin V Assay, Membrane, Flow Cytometry, Negative Control