Tissue was received in Optisol and explants plated within 24?hours. microscopy. As determined with Alamar Blue fluorescence, all concentrations of PI significantly decreased the number of cells AS-35 from all three preparation types compared with PBS. As determined Nedd4l by calcein/EH-1 viability test, mixed populations of cells and fibroblasts were less sensitive to PI treatment than goblet cells. All concentrations of PI, except for 0.25% used with goblet cells, substantially increased the number of dead cells for all cell populations. The H2O2 control also significantly decreased the number and viability of all three types of cells in both tests. Conclusion We conclude that PI, which is commonly used prior to ocular surgeries, is detrimental to human conjunctival stratified squamous cells, goblet cells and fibroblasts in culture. can be present.9 On the healthy ocular surface, these bacteria do not cause active infection due to the effects of multitude of antibacterial proteins secreted into the tears by the lacrimal gland, mucins synthesised and secreted by the cornea and conjunctiva and the blinking action of the lids.1 10 11 Despite these defence mechanisms, ocular infections do occur and are often attributable to trauma, disease or contact lens wear. Pathogenic bacteria have been identified on the ocular surface of patients with dry eye7 and infections from or can cause vision threatening bacterial keratitis and keratoconjunctivitis.12 The most common source of endophthalmitis-causing bacteria is the conjunctival and lid flora.13 14 Following surgical trauma, bacterial flora isolated from patients who developed endophthalmitis were identical to those isolated from the patients own conjunctiva and eyelid.15 To minimise the risk of infections during surgery or ocular injections such as anti-vascular endothelial cell growth factor (VEGF) therapies, ophthalmologists apply the antiseptic povidone iodine (PI) to the conjunctival sac prior to surgery. PI concentrations from 1% to 10% for between 30?s and 10?min reduce the number of bacterial colonies cultured from conjunctiva15C21 and the rate of endophthalmitis.15 22 The American Academy of Ophthalmology recommends a concentration of 5% PI to be applied prior to cataract surgery but does not recommend a specific duration or volume. Likewise, the European Society of Cataract and Refractive Surgeons recommends application of between 5% and 10% PI for no longer than 3?min but does not provide guidance on volume.23 There are, however, no published studies to date on the effect of PI application on the health of cells from the conjunctiva. The purpose of the present study was to determine in culture the effects of PI use on the viability of the three principal cell types present in the human conjunctiva. Materials and methods Materials RPMI, DMEM/F12 media, phosphate-buffered saline (PBS), HEPES, sodium AS-35 pyruvate, glutamine and penicillin/streptomycin were purchased from Lonza (Portsmouth, New Hampshire, USA). Fetal bovine serum was from Atlanta AS-35 Biologicals (Flowering Branch, Georgia, USA). Human serum, human insulin, Alamar Blue, calcein AM/ethidium homodimer-1 (EH-1) live/dead assay kit, AS-35 antibodies against cytokeratin 4 (CK4), cytokeratin 7 (CK7), anti-Ki-67 antibody and vimentin were provided by ThermoFisher (Waltham, Massachusetts, USA). Additional CK4 and CK7 antibodies were purchased from SantaCruz Biotechnology (Dallas, Texas, USA). PI solution (10%) was obtained from CVS (Woonsocket, Rhode Island, USA). Hydrogen peroxide, hydrocortisone, epidermal growth factor (EGF), fluorescein isothiocyanate (FITC)-conjugated lectin from Ulex europaeus agglutinin I (UEA) and lectin Bandeiraea Simplicifolia agglutinin conjugated to FITC were provided by Sigma-Aldrich (St Louis, Missouri, USA). MUC5AC antibody was purchased from Abcam (Cambridge, Massachusetts, USA). Secondary antibodies conjugated to Cy 2 or Cy 3 were.