eamag/protocols-with-steps huggingface dataset:
TITLE: intracellular calcium assay 
AUTHORS: Wakana Shoda, Naohiro Nomura, Fumiaki Ando, Hideaki Tagashira, Takahiro Iwamoto, Akihito Ohta, Kiyosih Isobe, Takayasu Mori, Koichiro Susa, Eisei Sohara, Tatemitsu Rai, Shinichi Uchida

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was used for the experiment conducted in " Sodium-calcium exchanger 1 is the key molecule for urinary potassium excretion against acute hyperkalemia"</div></div>

[STEPS]
?. Creation of Loading Buffer: solution A+ solution B■Solution A 5ml Recording Buffer 50µl Probenesid80μl Pluronic F-127 approximate 5 ml pure water (up to 10ml)■Solution B 50 µl DMSO50µℊ Fluo4Loading Buffer should be heated at 37 ℃
?. Cultured cells in glass bottom dish should  be washed with PBS, and replaced by 1.2 ml  loading buffer.Then, Incubate at 37 °C for 1 hour.   The drugs are added during this period.
?. Preparation for Recording Buffer while  cells are incubated in loading buffer. ■Recording Buffer5ml Recording Medium 50 μl   Probenesidapproximate 5ml pure water (up to 10ml)
?. After 1 hour incubation of loading buffer, the cells are  washed  with PBS ,  then replaced by 1.2 ml of recording buffer.
?. Following the administration of stimuli ( ex. 10mM KCl ), the fluorescence intensities of Fluo-4 are quantified from five regions of interest using LSM 510 Meta confocal microscopy and the ZEN 2009 software (Carl Zeiss, Oberkochen, Germany).
TITLE: A protocol for computerized quantitative analysis of nerve fibers, mast cells, enteric glial cells and the proximity of mast cells to the nerve fibers in 3D Images of human sigmoid mucosal biopsies
AUTHORS: Tao Li, Pu-Qing Yuan, Yvette Taché

[DESCRIPTION]
This protocol describes a step-by-step computational workflow that we developed by adapting Imaris 9.7-9.9 Surfaces Rendering Technology (https://imaris.oxinst.com/products/imaris-for-neuroscientists) to perform the quantitative analysis of nerve fibers, enteric glial and immune cells in 3D images of human sigmoid mucosal biopsies, as well as to assess the proximity of mast cells to the nerve fibers which cannot be easily portrayed and precisely measured with 2D images. The volumes of surface-masked nerve fibers and enteric glial cells, the numbers of spotted mast cells and the shortest distances of the centers of each individual spot to the surfaces of nerve fibers in 3D images were automatically computed and plotted correspondently with Imaris 9.7-9.9. This computerized protocol not only reduces the biases due to observer/examiner judgment and overcomes limitations of 2D images, but also much faster than measuring manually and allows us to quantitate a larger number of samples, increasing statistical accuracy. The created parameters also provide efficient reference for us to apply the New AI Machine Learning Segmentation implemented into Imaris 10.1 recently for our quantitative analysis of large datasets. This protocol has been using in out study relevant to the underlying peripheral mechanisms of visceral pain in irritable bowel syndrome.

[STEPS]
1. 
2.
TITLE: Flow Cytometry ECS Surface Antigens
AUTHORS: Michael Betts, Gregory Golden

[DESCRIPTION]
High-parameter flow cytometry enables identification and characterization of a wide range of cell populations within a biological sample. Analysis of cell-surface epitopes using a collection of fluorophore-labeled antibodies, or extracellular stain (ECS), sufficiently labels population-identifying markers for further analysis with flow cytometry. Importantly, ECS can be applied to cryo-preserved cell suspensions recovered in tissue culture media, enabling samples to be conveniently analyzed after collection and proper cryopreservation. However, consistent cryopreserved sample recovery and ECS procedures are critical to data comparison across multiple experiments. Herein, we describe a standardized protocol for cryopreserved sample recovery and ECS procedures for cell-surface epitope labeling. 

[STEPS]
SECTION: Procedure
1. Thawing and Resting

a. Pre-warm R10 media in a 37 °C water bath. 

b. Thaw samples in-vial using a 37 °C water bath.

c. Add thawed cells to 14 mL of R10, then spin cells at 500 xg for 5 min.

d. Resuspend cell pellet in 3 mL of R10 and count cells. 

e. Rest cells at least 3 hours (up to overnight) at 2x106 cells/mL in R10 medium + 1 µL/mL DNAse I at 37 °C , 5% CO².
Note: during resting, prepare  antibody cocktail master mix. Adjust volume of ECS for 50 µL per test with FACS buffer.

f. After resting, add PBS up to 15 mL or 50 mL (whichever is closer, rounding up) to cells and transfer to 15 mL or 50 mL conical tube.

g. Spin cells at 500 xg for 5 minutes at room temperature (RT).

h. Resuspend cells in PBS to 1x107 cells/mL and count. If cells are too dilute, re-spin cells and resuspend at 1x107 cells/mL. Transfer 200 mL of cells (2x106cells) into each well of a V-bottom 96 well plate.

SECTION: Procedure
2. Viability and extracellular staining (ECS)

a. Spin plate at 500 xg for 5  minutes at RT

b. Using a multichannel pipette, carefully remove the supernatant.

c. Using a multichannel pipette, add 45 µL of PBS to well.

d. Add 5 µL of 1:60 Aqua viability dye directly to cell pellet and resuspend cells.

e. Incubate for 10 minutes at RT in the dark.

f. Add 50 µL of ECS antibody cocktail to cells and incubate for 20 minutes at RT in the dark (prepare 1% PFA fixation buffer in the meantime).

g. Add 100 µL of FACS buffer to each well and spin plate at 500 xg for 5 minutes. 

h. Discard supernatant and fix cells with 200 µL of 1% PFA.

i. Run samples on flow cytometer.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 1">
<div>
<div>
<p>Synthetic biology holds great potential for addressing pressing challenges for mankind and our planet. One technical challenge in tapping into the full potential of synthetic biology is the low efficiency and low throughput of genetic transformation for many types of cells. In this paper, we discuss a novel microfluidic system for improving bacterial electrotransformation efficiency and throughput. Our microfluidic system is comprised of non-uniform constrictions in microchannels to facilitate high electric fields with relatively small applied voltages to induce electroporation. Additionally, the microfluidic device has regions of low electric field to assist in electrophoretic transport of nucleic acids into the cells. The device features hydro- dynamically controlled electric fields that allow cells to experience a time dependent electric field that is otherwise difficult to achieve using standard electronics. Results suggest that transformation efficiency can be increased by ∼4×, while throughput can increase by 100–1000× compared to traditional electroporation cuvettes. This work will enable high-throughput and high efficiency genetic transformation of microbes, fa- cilitating accelerated development of genetically engineered organisms.</p>
</div>
</div>
</div>

[STEPS]
?.
TITLE: 2X PEG 
AUTHORS: Jacquelina.Woods  

[DESCRIPTION]
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation. 

[STEPS]
3. Autoclave 121 °C15 min.

4. Store at 2-8 Room temperatureprotected from light. 

1. Dissolve components in ~700 mL deionized or ultrapure water. Heating to 50 °C is recommended to facilitate dissolution. 
2. Bring total volume up to  1 Lwith deionized or ultrapure water.

1.1. 200 g  , or equivalent 
1.2. 34.8 g,, or equivalent.
TITLE: Preventative at Home COVID-19 Based on Literature
AUTHORS: Rochelle Lieberstein Stern

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Abstract: Are there evidenced-based, reviews, articles of scientific validity, and/or clinical studies that </div><div class = "text-block">imply other actions, in addition to WHO, NIH, and CDC recommendations, which could help, safeguard, </div><div class = "text-block">or improve immunity response while home with or without COVID-19? This may include anything that </div><div class = "text-block">would be accessible via home items, mail online orders, food, or over-the-counter items in the drug </div><div class = "text-block">store. This review is exclusively based on scientific literature or medical professional observation</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Collaborator, Nathan VerBerkmoes (Oak Ridge National Labs), worked with Tucson Marine Phage Lab to develop more sensitive proteomics assays for viral isolates which we are now also using for environmental viral concentrates. A new sample prep method (FASP) was optimized to maximize the signal from our commonly low-protein containing samples, and then run using 2d-LC-MS-MS to maximize detection across the large dynamic range in isolate and environmental samples.

[GUIDELINES]
This method was optimized for bacteriophage by Kristen Corrier undergraduate research assistant at Oak Ridge National Laboratory (ORNL), Chemical Science Division using the commercial method from Protein Discovery (Knoxville, TN) as a starting point. <br /><br />This method was written by Kristen Corrier using the commercial method from Protein Discovery (Knoxville, TN) as a starting point. <br /><br />Dr. Nathan VerBerkmoes (ORNL, Chemical Science Division) oversaw the optimization process and creation of final method. <br /><br />This method is designed to prepare small quantities (~1-10&micro;g) of isolate bacteriophage or mixed bacterial/bacteriophage communities for shotgun proteomics via 2d-&shy;LC-&shy;MS/MS. <br /><br />Please note this method is not effective for large quantities of material (&gt;100&micro;g), standard solution digest should be used for such samples.&nbsp; The minimum protein amount needed is estimated to be 500ng but this is highly sample dependent. <br /><br />The main optimization of this method was to simplify it and greatly reduce the time required.&nbsp; This current method takes roughly half the amount of time (actually bench time) as the original commercial method, furthermore its now very straightforward to prepare 8, 16, or 24 samples at a time.

[STEPS]
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TITLE: Chimeric Protein-LAG and Peptostreptococcal protein L  sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA is used to study the interactions between protein-LAG (PLAG) and Peptostreptococcal protein-L (SpL) with different immunoglobulin preparations from mammalian and avian species.</div></div>

[STEPS]
?. This ELISA is used to study the interactions between protein-LAG (PLAG) and Peptostreptococcal protein-L (SpL) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of PLAG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and then, the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpL conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.28.
TITLE: Mitophagy induction using Oligomycin/Antimycin A
AUTHORS: Louise Uoselis

[DESCRIPTION]
Mitophagy induction in HeLa cells using Oligomycin/Antimycin A.

[STEPS]
SECTION: Day 1
1. Seed cells, aiming for a confluency of 80-90% at the time of treatment the next day. 
SECTION: Day 2
2. Feed cells for 1 h in an appropriate volume of standard growth media. 
SECTION: Day 2
3. To start the treatment, replace the media in each well with standard growth media that contains 10 uM Oligomycin, 4 uM Antimycin A, and 10 uM QVD. 
SECTION: Day 2
4. Harvest the samples after the desired treatment times.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Software pre-requisites:</p>
<p>Spades</p>
<p>SGA</p>
<p>cutadapt</p>
<p>trim_galore</p>
<p>Augustus</p>
<p>BUSCO</p>
<p> </p>
<p>Data sources:<br />  http://spades.bioinf.spbau.ru/spades_test_datasets/ecoli_sc/</p>
<p>  https://www.nature.com/articles/s41598-017-05436-4<br />Get from the ENA if possible.<br />  https://www.ebi.ac.uk/ena/data/view/PRJDB5352<br />  https://www.ncbi.nlm.nih.gov/bioproject/PRJDB5352</p>
<p>E.coli reference<br />  https://www.ncbi.nlm.nih.gov/nuccore/NC_000913</p>
<p> </p>

[STEPS]
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TITLE: U Mass - Lipid metabolism
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block"><span>
Lipid metabolism is estimated by measuring systemic clearance of [1-</span><span style = "vertical-align:super;">14</span><span>C] palmitate following a bolus injection in awake mice. Lipid metabolism is altered in obese mice.</span></div></div>

[STEPS]
?. Survival surgery is performed to establish a chronic indwelling catheter at 5~6 days prior to experiment for intravenous infusion. (refer to M1023: Surgery-jugular vein cannulation)
?. Mice are fasted overnight (~15 hours) or for 5 hours prior to the start of experiment.
?. Place a mouse in a rat-size restrainer with its tail tape-tethered at one end.
?. Expose and flush the intravenous catheter using saline solution. Then, connect the catheter to the CMA Microdialysis infusion pump.
?. Collect plasma sample (10 µl) before the start of experiment (basal-0 min) to measure basal glucose levels.
?. Administer a bolus intravenous injection of 20 µCi of [1-14C] palmitate to start the experiment.
?. Rapidly collect plasma samples (10 µl each) at 0.5, 1, 2, 3, 4, and 5 min after injection to measure systemic [1-14C] palmitate concentrations.
?. At the end of experiment, mice are euthanized, and tissues may be collected for further studies.
?. For data analysis, plasma [1-14C] palmitate levels vs. time after palmitate injection are plotted, and area-under-curve may be calculated to estimate systemic clearance of labeled-palmitate and lipid metabolism.
TITLE: Immunoblotting (Western) Protocol
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the western blot technique.


[STEPS]
SECTION: Immunoblotting (Western)
1. Run protein lysates using desired Criterion™ TGX™ gels.
SECTION: Immunoblotting (Western)
2. Transfer onto nitrocellulose membranes.
SECTION: Immunoblotting (Western)
3. Detect protein of interest using appropriate primary antibodies at 4 °C 60 min, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min at Room temperature.

SECTION: Immunoblotting (Western)
4. To visualize, use chemiluminescent substrates and image using ImageQuant LAS 4010 detection system. 
SECTION: Immunoblotting (Western)
5. Perform densitometry on Western blot images using ImageQuant TL 8.1 software.
TITLE: Apple Keto Gummies Australia
AUTHORS: soufianeelbekri  

[DESCRIPTION]
Quite possibly the most well known ways for individual to get in shape and cut back excess is the keto diet. Today, we will enlighten you concerning Apple Keto Gummies Australia diet pills. This new 

[STEPS]
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TITLE: Data Analysis Procedures
AUTHORS: Deziray.Howard

[DESCRIPTION]
Draft of the procedures and workflow for data analysis within PDI

[STEPS]
SECTION: Introduction
1. Welcome to the PDI Data Analysis playbook

This document serves as a comprehensive guide to the processes and methodologies followed by our team in data analysis to derive meaningful insights and support informed decision-making. We aim to ensure consistency, reliability, and efficiency in our analytical endeavors by adhering to these standardized procedures.

Purpose

This manual is designed to provide a clear and structured framework for conducting data analysis within our team. It outlines the key steps, best practices, and responsibilities associated with data collection, cleaning, exploratory analysis, statistical modeling, reporting, and more. Whether you are a seasoned analyst or are new to our team, this manual is a valuable resource to help you navigate our data analysis processes.
SECTION: Data Collection
3. 
SECTION: Governance and Compliance
2. Compliance with FedRAMP

Ensure that all data analysis and transferal programs adhere to FedRAMP (Federal Risk and Authorization Management Program) standards. Verify that the selected tools and platforms have obtained FedRAMP approval. 


Endpoint Security

Ensure that antivirus software and security patches are up to date. Ensure that government-issued equipment is used when necessary. If teleworking, only work on secured and private networks (do NOT conduct work on public Wi-Fi, such as Starbucks). Refer to USDA Information Security Awareness, USDA policies, and system-specific rules.

SECTION: Data Cleaning
4. 
SECTION: Exploration and Analysis
5. 
SECTION: Data Modeling
6. TBD
TITLE: Emotional affection on a sustained attention task
AUTHORS: Luis Pinel, Miguel A. Perez-Nieto, Marta Redondo, Luis Rodríguez-Rodríguez, Fernando Gordillo, Leticia L. Mateos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol includes the database used in this study. You will find more details about how it was used and the results of the study in the published article.</div></div>

[STEPS]
TITLE: Preparing phytoplankton samples for elemental carbon and nitrogen analysis
AUTHORS: Jana Hinners, Sinead Collins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for the preparation of phytoplankton samples for elemental carbon and nitrogen analyses.</div></div>

[STEPS]
?. [Preparing GFF filters]
Wrap up to 20 Whatman GFF filters in aluminium foil and precombust them for 6 hours at 450degrees C, store filters airtight afterwards
?. [Preparing GFF filters]
Weigh precombusted filters and store labelled and individually (e.g. in 6-well microtitre plates)
?. [Preparing phytoplankton cultures]
Grow 45 mL phytoplankton cultures in 50 mL flasks until they reach mid-exponential phase, culture are dense enough when some colour is visible
?. [Applying samples onto filters]
1 mL used of each sample used for cell counting (e.g. with fluorescence plate reader/FlowCam/FlowCytometry?Microscopy)
?. [Applying samples onto filters]
Use a vacuum filtration unit to apply the phytoplankton samples onto the GFF filtersFrom the remaining 44 mL per sample, 20 mL each used for 2 replicate filtersApply the ample onto filter using a pipette, carefully check volumeWash each the edge of the filter unit and the filter with 10mL dest. water
?. [Applying samples onto filters]
Filters with biomass can be stored again in the 6-well plates,Let filters dry with loose lid overnight
?. [Preparation of filters for elemental analysis]
Dry filters at 40 degrees C for 2-3 daysWeigh all dried filters again and calculate the net weight of the dry biomass for each filter
?. [Preparation of filters for elemental analysis]
For work with an elemental analyzer that measures total carbon content, the inorganic carbon content has to be removed: (this can take place in the 6-well platesSoak filters with 200uL dest. water, then apply 100uL 2M HCl onto each filter to remove any inorganic carbon from the filtersLet filters dry overnight
?. [Preparation of filters for elemental analysis]
Filters are now ready for elemental analysis
TITLE: Quantification of IHC Using an Inverted Confocal Microscope and NIS-Elements Program-Killinger Lab 2024
AUTHORS: Solji Choi

[DESCRIPTION]
Protocol to quantify DAB stained sections using NIS-Elements.

[STEPS]
1.       
Capture brightfield images with an inverted
confocal microscope with a 20X objective (Nikon A1R). 
2. Conduct annotation of each tissue section within a bounding box of 2000×2000 pixels for
mouse tissues and 2863×2454 pixels for human tissues. 
3.       Use a manual RGB-based color thresholding
algorithm for mouse tissues to exclude pure methyl green nuclei staining from
the measurement.  For human tissues, use NIS-elements
(version 5.10.01,
https://www.microscope.healthcare.nikon.com/products/software/nis-elements,
RRID:SCR_014329) auto-thresholding algorithm.
4.       
Export the percentage area of the thresholded
signal (object area fraction provided in the program) and normalize to the average
value of non-CIAP treated tissues.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
This protocol comes from a group of other protocols.This protocol is (3) of (4): <br />1. <a href="https://www.protocols.io/view/Large-Volume-Marine-Cyanophage-Phage-Protocols-c3iykd" target="_blank">'Large Volume Marine Cyanophage Phage Protocols'</a><br />2. <a href="https://www.protocols.io/view/DNA-Extraction-Protocol-c32yqd" target="_blank">'DNA Extraction Protocol'</a><br />3. <a href="https://www.protocols.io/view/DNA-Precipitation-Protocol-c34yqv" target="_blank">'DNA Precipitation Protocol'</a><br />4. <a href="https://www.protocols.io/view/Checking-DNA-Concentration-with-Agarose-Gel-c35yq5" target="_blank">'Checking DNA Concentration with Agarose Gel'<br /><br /></a>Needed:<br />
<ul>
<li>Ethanol</li>
<li>Ice</li>
<li>Microfuge</li>
<li>70% ethanol made from 100% with autoclaved milli-Q-water)</li>
<li>Centrifuge</li>
<li>TE Buffer</li>
</ul>

[STEPS]
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