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A preprint repository for life science protocols
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Maintenance of Drosophila S2 cells and CO2 exposure
Q&A Nov 24, 2025Maintenance of Drosophila S2 Cells and CO2 ExposureCell culture conditionsDrosophila S2 cells were maintained at room temperature and protected from light in Schneider’s Insect Medium supplemented with 10% fetal bovine serum (FBS; Valley Biomedical) and 0.2% Penicillin-Streptomycin (Gibco). Cells were cultured under standard non-CO2 incubator conditions. Plate preparation for cell attachmentSix-well plates were pre-treated to promote S2 cell attachment. Wells were incubated with 1 N HCl for 1 h ...
In vitro culture of adult mouse DRG neurons
Q&A Nov 24, 2025This protocol is a detailed description of the method used in the research article Multimodal control of dendritic cell functions by nociceptors, published in Science, 2023. 379, eabm5658 Materials and reagentsPoly-D-LysineLamininPBSLeibowitz 15 mediumPercoll Digestion solution 1 (filter-sterilized): 60U papain 0.5 mM EDTA 1.5 mM CaCl2 in HEPES-buffered HBSS Digestion solution 2 (filter-sterilized): 4mg/ml Collagenase type-2 5mg/...
Cryopreservation
Q&A Nov 13, 2025Cryopreservation and sample preparation Rationale: Samples need to be dehydrated before being embedded in a resin. However, classic dehydration techniques will result in the loss of DMSP from the cells. To effectively prevent the loss of DMSP from the cells, the entire sampling procedure leading to NanoSIMS analysis must be water-free, with all steps performed under strict anhydrous conditions. For this, we use cryopreservation techniques followed by freeze substitution in an acrolein-ether mixt...
Modeling and computational docking
Q&A Nov 12, 20251. Docking methodA homology model of PfcIRS (Q8IDZ9; PF3D7_1332900) was created using SccIRS (7d5c.1) as a template using Swiss-Model (79). The SccIRS structure is bound by RM-A and an ILE-AMP intermediate located within the catalytic site of the Rossman fold and importantly, no tRNA is bound for this published structure. To help position tRNA within the catalytic domain in our homology model, we used the structure of EcLRS (4AQ7). Features such as the N-terminal catalytic Rossman fold domain...
2.5. Stool Bacterial Extracellular Vesicle Extraction and Removal of Exosomes
Q&A Nov 10, 2025Isolation of Vesicles from Stool SamplesModified by Chanel Mosby from: Wu, J. et al. A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS. J. Proteomics Bioinform. 12, 38–42 (2019). Materials25 mg of mice stoolPBS, dPBSdPBS + 1X Protease inhibitor cocktailUltra centrifuge tubes for the small swinging rotor3 screw-cap 2 ml tubesRecommended to label as: pellet, S/N, bEV Procedure---benchtop ---Weigh out 25 mg of stool into a screw-top 2 ml tube la...
Histochemical GUS analysis in transgenic rice
Q&A Nov 7, 2025Bio-Protocol ExchangeConcise Method Switchgrass (Panicum virgatum L.) Promoters for Green Tissue-Specific Expression of the MYB4 Transcription Factor for Reduced-recalcitrance Transgenic Switchgrass Wusheng Liu1,2†, Mitra Mazarei1,6†, Rongjian Ye1†, Yanhui Peng1, Yuanhua Shao1, Holly L. Baxter1,6, Robert W. Sykes3,6, Geoffrey B. Turner3,6, Mark F. Davis3,6, Zeng‑Yu Wang4,6, Richard A. Dixon5,6 and C. Neal Stewart Jr.1,6* Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA.D...
Organoid Seeding Protocol on Chips for MEA recordings
Q&A Oct 26, 2025Organoid Seeding Protocol on Chips MEA Chip Preparation Please refer to the supplier’s protocol for chip surface preparation.For MaxOne single-well chips (Maxwell Biosystems), the preparation includes:Cleaning with 1% Terg-a-zyme solution for a minimum of 2 hours at room temperature.Washing thoroughly with distilled water.Sterilizing with 70% ethanol.Drying under laminar airflow in a sterile cell culture hood.Poly-L-Ornithine Hydrobromide Coating Dissolve Poly-L-Ornithine hydrobromide (Sigma P36...
Sample preparation for light sheet microscopy
Q&A Oct 17, 2025I have experience with intravital imaging using the Zeiss Lightsheet Z.1 with zebrafish embryos and larvae at early developmental stages. In addition, I have also performed intravital imaging of living killifish, using a transparent line as described in the respective publication. For all experiments, I used the standard chamber (there is no extra-large chamber available, and it would not fit into the setup anyway), which means that the samples need to be small enough to fit into the chamber. Th...
From Allelic Heterogeneity to Biomechanical Phenotypes: A Functional New Genomic Model of ABCC11 Polymorphisms and their Role in Human Chemical Ecology
Q&A Oct 13, 2025AbstractThe ABCC11 gene, through a single nucleotide polymorphism (SNP; rs17822931), is established as a primary determinant of human axillary secretion. Such a composition dictates the presence of both a binary phenotype of earwax (dry/wet) and the absence or presence thereof of characteristic body odor. However, significant phenotypic heterogeneity exists within genotypic groups, suggesting a more complex genetic architecture. This paper proposes a functional genomic model that expands upon t...
Fluorescence-based ion transport assays using Proteoliposomes
Q&A Sep 20, 2025Abstract Divalent metal ion transporters are conserved across all domains of life and play essential roles in diverse processes such as manganese acquisition during nutritional immunity in bacteria [1] and iron homeostasis in higher eukaryotes [2], [3]. Traditional techniques, such as electrophysiological assays, are often unsuitable due to the slow kinetics of many membrane transporters. To overcome these limitations and to investigate both the activity and ion selectivity of transporters, also...