Isolation and Characterization of Primary Retinal Microglia From the Human Post-mortem Eyes for Future Studies of Ocular Diseases

Microglia, the primary resident immunocytes in the retina, continuously function as immune system supervisors in sustaining intraocular homeostasis. Microglia relate to many diseases, such as diabetic retinopathy, glaucoma, and optic nerve injury. To further investigate their morphology and functions in vitro, a reliable culture procedure of primary human retinal microglia is necessary. However, the culture condition of microglia from the adult retina is unclear. Researchers created several protocols, but most of them were carried out on rodents and newborns. This study describes a protocol to isolate and characterize human primary retinal microglia from human post-mortem eyes. The whole procedure started with removing the retinal vessels, mechanical separation and enzymatic dissociation, filtration, and centrifugation. Then, we cultured the cell suspensions on a T-75 flask for 18 days and then shook retinal microglia from other retinal cells. We found numerous retinal microglia grow and attach to Müller cells 10 days after seeding and increase rapidly on days 14–18. Iba1 and P2RY12 were used to qualify microglia through immunofluorescence. Moreover, CD11b and P2RY12 were positive in flow cytometry, which helps to discriminate microglia from other cells and macrophages. We also observed a robust response of retinal microglia in lipopolysaccharide (LPS) treatment with proinflammatory cytokines. In conclusion, this study provides an effective way to isolate and culture retinal microglia from adult human eyes, which may be critical for future functional investigations.

Automated hiPSC culture and sample preparation for 3D live cell microscopy

Our goal is to identify and understand cellular behaviors using 3D live imaging of cell organization. To do this, we image human inducible pluripotent stem cell (hiPSC) lines expressing fluorescently tagged protein representing specific cellular organelles and structures. To produce large numbers of standardized cell images, we developed an automated hiPSC culture procedure, to maintain, passage and Matrigel coat 6-well plastic plates and 96-well glass plates compatible with high-resolution 3D microscopy. Here we describe this system including optimization procedures and specific values for plate movement, angle of tips, speed of aspiration and dispense, seeding strategies and timing of every step. We validated this approach through a side-by-side comparison of quality control results obtained from manual and automated methods. Additionally, we developed an automated image-based colony segmentation and feature extraction pipeline to predict cell count and select wells with consistent morphology for high resolution 3D microscopy.

Matrix Extension Study: Listeria Right Now™ Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment: AOAC Performance Tested MethodSM 081802

Background: Listeria Right Now™ is a novel, enrichment-free test for the detection of Listeria spp. in swab samples taken from environmental surfaces. Results are available in less than 1 h. In a previous Performance Tested MethodSM (PTM) study, the test was validated for swab samples from stainless-steel and sealed concrete surfaces. Objective: A PTM matrix extension study was conducted to validate the method for the detection of Listeria spp. in swab samples from ceramic tile, plastic, and rubber surfaces. Methods: Performance of the Listeria Right Now method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedure for the detection of Listeria spp. in swab samples taken from inoculated ceramic tile, plastic, and rubber surfaces. Data were analyzed using a probability of detection model. Results: There were no significant differences in performance between the Listeria Right Now and reference culture methods for any of the three surfaces tested, as determined by probability of detection analysis. Conclusions: The Listeria Right Now method is an effective procedure for the detection of Listeria spp. from a variety of environmental surfaces. Highlights: Listeria Right Now provides accurate results, without enrichment, in real time. This enables food industry personnel to react swiftly to suspected Listeria contamination incidents.

Effect of EDTA and QMIX Ultrasonic Activation on the Reduction of Microorganisms and Endotoxins in Ex Vivo Human Root Canals

The present study aimed to compare the effectiveness of QMiX and 17% EDTA associated to passive ultrasonic irrigation (PUI) or manual agitation (MA) on the reduction of E. faecalis, E. coli and LPS from root canals. Forty single rooted human teeth were randomly divided into four groups (n=10), according to the final irrigation protocol: EDTA+MA, QMiX+MA, EDTA+PUI, QMiX+PUI. Sample collections were obtained from the root canal content immediately before preparation (baseline-S1), after instrumentation (S2), after final irrigation protocol (S3) and 7 days after instrumentation and final irrigation (S4). The antimicrobial effectivity and on endotoxin content were analyzed by culture procedure (CFU/mL) and LAL assay (EU/mL), respectively. The results were statistically analyzed by Kruskal-Wallis and Friedman test (α=5%). QMiX+MA and QMiX+PUI reduced 100% of E. coli and E. faecalis bacteria and also prevented E. faecalisregrowth at S4. EDTA significantly reduced E. coli, but it was not effective in reducing E. faecalis. All protocols reduced EU/mL when compared to S1, however at S4 there was a significant reduction of EU/mL only in the QMiX+MA and QMiX+PUI groups in relation to S3 and S2, respectively. Final irrigation with QMiX associated with MA or PUI had superior antibacterial efficacy compared to EDTA, eliminating 100% of E. coli and E. faecalis strains. In addition, QMiX+PUI reduced 97.61% of the initial content of LPS.