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A preprint repository for life science protocols
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Optimized ex vivo preservation and FACS isolation of Drosophila ovarian germline and follicle cells for cell-type-specific gene-expression analysis
Q&A Published: Sep 18, 2026Optimized ex vivo preservation and FACS isolation of Drosophila ovarian germline and follicle cells for cell-type-specific gene-expression analysisRounab Sarkar¹, Sautan Show¹, Anjali Mohan¹, Upendra Nongthomba¹*1 Department of Developmental Biology and Genetics; Indian Institute of Science; Bengaluru – 560012, India.* Corresponding author: Prof. Upendra Nongthomba, upendra@iisc.ac.inAbstract:Cell-type-specific molecular profiling of the Drosophila melanogaster ovary is important for understandi...
Cell Surface CD74 Measurements.
Q&A Published: Sep 16, 2026Cell Surface CD74 Flow Cytometry ProtocolRequired MaterialsReagents:Thawing/Stim Medium: RPMI 1640 medium supplemented with 5% FBS, 1% Glycine (2 mL of 100× stock), and 1% Pyruvate (2 mL of 100× stock)Staining Solution: 1× PBS supplemented with 5% BSAAntibodies/Dyes:FITC-conjugated anti-CD74 (Clone: MB731; BD Biosciences)APC-conjugated anti-CD11b (Clone: ICRF44; BioLegend)7-aminoactinomycin D (7-AAD; BD Biosciences)Equipment: Water bath (37°C), Centrifuge, Accuri C6 flow cytometer (BD), 96-well ...
2.5. MitoSox red staining
Q&A Published: Sep 15, 2026We recommend optimizing the procedure for dissociated mouse hepatocyte spheroids, as the dissociation process itself may affect mitochondrial ROS levels and cell viability.MitoSOX stock preparation and working concentrationMitoSOX Red (Thermo Fisher Scientific) was dissolved in DMSO to prepare a 5 mM stock solution. Immediately before use, the stock was diluted in pre-warmed DPBS to a final working concentration of 5 μM. Stock aliquots were protected from light and repeated freeze–thaw cycles we...
A fluorescent-based high-throughput method to quantitatively measuring PSS1-mediated phosphatidylserine synthesis
Q&A Published: Sep 14, 2026Abstract:With phosphatidylcholine and L-serine, phosphatidylserine synthase 1 (PSS1) synthesizes phosphatidylserine, a phospholipid that is a key component in cell membranes. Here, we provide a step-by-step protocol examining the enzymatic acidity of PSS1 in vitro. This in vitro assay quantitatively measures Choline, a byproduct generated in the biochemical reaction, featuring simple operation, high sensitivity and low cost without the requirement for radioactive labeling. It enables rapid eva...
Microalgae Morphology
Q&A Published: Sep 14, 2026For morphology analysis, microalgae samples were diluted in TAP media to a final density of 1×106 microalgae/mL, then 10 µL were loaded onto a glass slide and taken to the light microscope. Morphology was evaluated by optical microscopy (Dmi1 microscope, Leica), with 10x/0.4 and 40x/0.65 objectives and imaged with a standard MS60 digital camera (Mshot). Cell area quantification was done on 40x images with ImageJ, where a background threshold was applied and the total area of each separate micro...
Cell culture and calcium switch assay
Q&A Published: Sep 9, 2026Calcium Switch Assay for Synchronized Junction Disassembly and Reassembly in Human Intestinal Epithelial MonolayersArturo Raya-Sandino and Asma Nusrat*Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USAFor correspondence: anusrat@umich.eduAbstractThe calcium switch assay provides a synchronized system for examining the disassembly and reassembly of calcium-dependent epithelial cell-cell junctions1-3. Here, we describe a protocol for evaluating the recruitment of th...
Stimulated emission depletion microscopy (STED)
Q&A Published: Sep 7, 2026See attached protocol file (better numbering) Immunohistochemistry for STED on free-floating vibratome sections Prior to immunohistochemical staining, the animals were perfused with 1xPBS and heparin (20 U/ml), followed by 4% PFA, and the brains were removed from the skull and post-fixed overnight in 4% PFA. Tissues were stored in 1xPBS at 4ºC and sectioned into 50 µm thick sections using the HM650V Vibratome (Thermo Fisher Scientific). For long-term storage, use sodium-azide (NAN3). Sections w...
Site-Specific L-Anap Incorporation by Genetic Code Expansion for Minimally Perturbative Imaging of TDP-43 in Mammalian Cells
Q&A Published: Sep 3, 2026Abstract Fluorescent protein tags are widely used to investigate protein localization and dynamics, but their relatively large size can perturb protein structure and function. In contrast, genetic code expansion offers a minimally disruptive alternative by enabling site-specific incorporation of non-canonical amino acids, such as L-Anap (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid). Here, we describe a protocol for labeling TDP-43 with the intrinsically fluorescent non-canonical amino...
Fusion inhibition assay using split GFP
Q&A Published: Sep 2, 2026Split GFP cell-cell fusion assayDay 1: seeding HEK293T cellsIn 96 well plate: cells will be transfected with GFP11 and will not be detachedIn 6 well plate: cells will be transfected with Spike + GFP1-10 and will be detached + added to cells in 96 well plate- Cell seeding in 96 well plates: 8000 cells/well, to transfect the next day. If transfecting on day 3: seed 4000 cells/well- Cell seeding in 6 well plates: 200 000 cells/well, to transfect the next day. If transfecti...
Leaf Rolling, Leaf Drying, and Drought Recovery Scores
Q&A Published: Sep 1, 2026Field scoring of rice leaf rolling, leaf drying and recovery under reproductive-stage droughtMethod used in the rainout-shelter experiments reported by Ghneim-Herrera et al. (2017), with visual scores based on the IRRI Standard Evaluation System for Rice (SES). ScopeThis protocol describes the field procedure used to follow leaf rolling (LR), leaf drying (LD) and drought recovery (DRR) in rice exposed to water deficit at the reproductive stage. The observations are non-destructive and are made o...