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A preprint repository for life science protocols
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Transposon analysis from RNA sequencing
Q&A Published: Oct 7, 2026Paired-end reads were filtered and trimmed using fastp (version 0.21.0 with default parameters) (1). Reads with quality inferior to 20 were removed from the data setsData sets were aligned against the Arabidopsis thaliana genome (TAIR10) using STAR (version 2.7.2a) allowing two mismatches (--outFilterMismatchNmax 2).Transposable elements (TEs) were defined according to Panda et al, 2016 (2). We used their TE annotation for our analysis (https://github.com/KaushikPanda1/AthalianaTETranscripts)Usi...
Protocol for assessing decision-making in rats using a low-cost rodent Iowa Gambling Task
Q&A Published: Oct 6, 2026Protocol for assessing decision-making in rats using a low-cost rodent Iowa Gambling Task Jyotsna Pandey1*, Sakshi Sharma1*, Varsha Singh2, Suman Jain3* Shared First authorship1 School of Interdisciplinary Research, Indian Institute of Technology, Delhi2 Humanities and Social Sciences, Indian Institute of Technology, Delhi3 Department of Physiology, All India Institute of Medical Sciences, New Delhi Abstract Decision-making is critical for survival, involving assessments of potential rewards an...
2.6. In Vitro Antidiabetic Activity (α-Amylase Inhibition Assay)
Q&A Published: Oct 5, 2026α-Amylase Inhibition Assay (Starch-Iodine Method)Mohammed Abu Tayab1’ *1 Department of Pharmacy, International Islamic University Chittagong, Chattogram 4318, BangladeshCorresponding author: abutayab.me@gmail.com Abstractα-amylase inhibition assay is an in vitro test for determining the potential antidiabetic activity of plant extracts and other substances, using acarbose as a standard inhibitor. The assay, known as the starch-iodine method, measures the remaining starch after incubation with α-...
PK/LDH Coupled Assay.
Q&A Published: Oct 5, 2026Pyruvate kinase/lactate dehydrogenase coupled assay Overview:This is a coupled enzyme assay that indirectly measures ATP consumption by coupling the ADP produced to NADH oxidation via:Pyruvate Kinase (PK): converts ADP + PEP → Pyruvate + ATPLactate Dehydrogenase (LDH): converts Pyruvate + NADH → Lactate + NAD⁺The decrease in NADH absorbance (typically at 340 nm) or fluorescence (excitation ~340 nm, emission ~460 nm) reflects ATP consumption by the kinase of interest (e.g., Btk). Stock Solutions ...
Estimating phycobilisome coupling to photosystems I and II and the PSII:PSI ratio in cyanobacteria using low-temperature (77 K) fluorescence spectroscopy
Q&A Published: Oct 2, 2026Estimating phycobilisome coupling to photosystems I and II and the PSII:PSI ratio in cyanobacteria using low-temperature (77 K) fluorescence spectroscopy Tomáš Zavřel1, Anna Segečová1, Mariann Kis2, Eva Kotabová3, Ondřej Prášil3, Jan Červený1, Gábor Bernát2 1Department of Adaptive Biotechnologies, Global Change Research Institute, Czech Academy of Sciences, Brno, Czechia2HUN-REN Balaton Limnological Research Institute, Tihany, Hungary 3Centre Algatech, Institute of Microbiology of the Czech Ac...
Immunofluorescence with J2 antibody
Q&A Published: Oct 1, 2026J2 antibody immunofluorescence Cell Plating and Knockdown:Place 12mm circular cover slips into a 24-well plateUV sterilize for 10minWash once with DMEM mediaHarvest HeLa cells and countDilute cells to 40K/ml and plate 1ml of cells per wellDuring trypsinization, mix:50ul of OptiMEM with 1ul of RNAimax per sample (Tube A)Incubate at RT for 5 min50ul of OptiMEM with 1ul of Control or SETX siRNA per respective samples (Tube B)After 5min, add Tube A to Tube B, mix and incubate at RT for 20 minSplit T...
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...