Q&A Feb 5, 2026

Determination of BV2 Microglial Phagocytic Activity (Fluorescent Microspheres / FluoSpheres Assay; Flow Cytometry/Microscopy) OverviewThis assay quantifies phagocytosis of fluorescent carboxylate-modified microspheres by BV2 microglia. After exposure to the test compound, cells are incubated with FluoSpheres, washed to remove non-internalized beads, detached with Accutase containing Hoechst 33342 to identify nuclei, and analyzed by flow cytometry. Phagocytosis is reported as the percentage of Fl...

Q&A Jan 31, 2026

Lentiviral Packaging, Concentration, and Transduction  Introduction: This protocol describes the production, concentration, and usage of second generation VSV-G pseudo typed lentiviruses for gene delivery in mammalian tissue culture. Growing Packaging Cells: Grow 293T viral packaging cells in DMEM containing 4.5 g/L glucose and 110 mg/mL sodium pyruvate, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. Passage 90% confluent cultures by trypsinization and re-seed 1:5 or ...

Q&A Jan 28, 2026

Immunofluorescence ProtocolQuintana lab Rinse sections of antifreeze solution with PBS pH 7.4........................…………………………................. 3x5’Block sections with PBS 0.2% Triton X-100 + 10% normal donkey serum (NDS) (9ml +1ml) ......…....... 1h at RTIncubate with primary antibody in PBS + 0.2% Triton X-100 + 1% NDS (9.9ml +0.1ml)...........…............ ON at 4ºCWash with PBS 0.2 Triton X-100.....................…………………………………………………….................. 3x5’Incubate with secondary antibody P...

Q&A Jan 26, 2026

Co-immunoprecipitation (co-IP) Following SUMOylation/Ubiquitination Perturbation(TAK-981/TAK-243 treatment with NEM-preserved, non-denaturing lysis)OverviewThis protocol describes the workflow for treating cells with TAK-243 (ubiquitin E1/UBA1 inhibitor) and/or TAK-981 (SUMO E1 inhibitor), harvesting cells under cold conditions, preparing NEM-preserved non-denaturing lysates, and performing co-immunoprecipitation (co-IP) for downstream immunoblot analysis. Because SUMO conjugates can be lost dur...

Q&A Jan 23, 2026

Bulk RNA-sequencing Differential Gene Expression Generation: With respect to bulk RNA-sequencing analysis, differentially expressed genes with respect to condition (pregnant/pseudopregnant/superovulation) and time (0.5 days-post-coitus (dpc) pregnant vs 1.5 dpc pregnant) were generated utilizing the Biojupies web-interface pipeline (https://maayanlab.cloud/biojupies/). Excel sheets containing differentially expressed genes were exported from Biojupies, and further manipulated with respect to Log...

Q&A Jan 14, 2026

Protocol for GSEA/GOTake the mean of forward and reverse experiments.Rank the list and submit to GSEA (Gene set Enrichment Analysis) against Gene-Ontology (GO) database. This software is free to download online and easy to use.Since we pre-ranked list use ‘Run GSEAPreranked’. Requires a list in .rnk format. This reflects the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genesControl is a permutated dataset (1000 iterations)40 GO terms passed the threshold...

Q&A Jan 9, 2026

Setting up Drosophila primary cultures from RasV12-expressing genotypesThis is a detailed protocol for establishing primary cultures that has more details than the original method in a paper describing derviation of continuous lines from Drosophila culures (https://elifesciences.org/articles/85814#s4). Author for enquiries: Amanda Simcox, Department of Molecular Genetics, Ohio State University, Columbus OH 43210. simcox.1@osu.edu Materials and reagents:Egg laying medium: Separately autoclave sol...

Q&A Jan 2, 2026

The calculation of blood oxygen saturation (StO2) is based on the modified Beer–Lambert law applied at two wavelengths (660 nm and 850 nm). Briefly, the measured optical attenuation at each wavelength is related to the concentrations of oxyhemoglobin (C_Ox) and deoxyhemoglobin (C_Deox) through their wavelength-dependent extinction coefficients. 1.Known parametersWavelengths: λ1 = 660 nm and λ2 = 850 nm.Effective path length: d_eff (accounts for geometric separation and the differential pathlengt...

Q&A Dec 26, 2025

 Hayao Ohno1, 2, *, Takanori Ida3, 4, *, and Yuichi Iino5  1Division of Material and Biological Sciences, Graduate School of Science, Japan Women's University, Tokyo, Japan2Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Tokyo, Japan3Department of Veterinary Physiology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan4Center for Animal Disease Control, University of Miyazaki, Miyazaki, Japan5Department of Biological Sciences, S...

Q&A Dec 24, 2025

Biological Electron Microscopy Sample Preparation Protocol for Rabbit Corneal Tissue1. Animal Tissue Sample Collection:New Zealand rabbits were euthanized, and corneal tissues from different groups were collected. Sampling was performed within 1-3 minutes post-euthanasia. Corneal tissue samples were trimmed to approximately 2mm × 2mm in size and made as thin as possible. If immediate trimming was not feasible, samples were first placed in electron microscopy fixative for approximately half an ho...