eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Human tyrosinase, a protein involved in the melanogenesis pathway, has various mutations in its corresponding gene <em>(TYR)</em> which have been linked to Oculocutaneous Albinism type 1 (OCA1), an autosomal recessive disease. Naturally, this inherited disorder leads to decreased melanocyte pigmentation accompanied by various visual dysfunction. Recombinant human tyrosinase was individually overexpressed in whole <em>Trichoplusia ni</em> (<em>T. ni</em>) larvae. Purification of catalytically active protein was achieved through immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Moreover, confirmation of protein identity was accomplished using human tyrosinase-specific antibodies and mass spectrometry. Lastly, the activity for both the intra-melanosomal domain (truncated, hTyrC<sub>t</sub><sub>r</sub>) and full length (hTyr) protein was displayed through L-DOPA and L-Tyrosine conversion into dopachrome (measured at 475 nm). This method of protein purification allows for higher yield recombinant hTyr and hTyrC<sub>t</sub><sub>r</sub> protein from whole insect biomass to further elucidate characterization of both structure and role in human melanogenesis with the overall goal to use hTyr for enzyme replacement therapy.</p>

[BEFORE_START]
<p>Table 1. Reagents and Solutions</p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/vfjinhe.png" data-src="https://s3.amazonaws.com/pr-journal/vfiinhe.png" data-ofn="Picture1.png" /></p>
<p>* To diminish chances of air introduction all buffers should be freshly prepared, filtered and degassed before connection to chromatography system. </p>
<p>** Non-ionic detergents: Triton X-100 or hydrogenated Triton X-100 (hTriton X-100).</p>
<p>**Truncated recombinant human tyrosinase (hTyrC<sub>t</sub><sub>r</sub>) does not require detergent. </p>

[GUIDELINES]
<p style="text-align: left;"><strong>Figure 1. Schematic Overview of Tyrosinase Purification</strong></p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/u47inhe.png" data-src="https://s3.amazonaws.com/pr-journal/u46inhe.png" data-ofn="Picture1.png" /></p>
<p><span style="font-weight: 400;">Frozen </span><em><span style="font-weight: 400;">T. ni </span></em><span style="font-weight: 400;">larvae were used for tyrosinase protein purification.The larvae were homogenized (see Materials and Steps sections) then eluted through an IMAC column, dialyzed, and subsequently underwent size exclusion chromatography through two columns (Sephacryl S200 HR 16/60 &amp; Superose 12 10/300 or Sephacryl S300 HR 16/60 &amp; Superdex 200 Increase 10/300) followed by an IMAC (Ni-NTA Resin Gravity Flow) polishing step. Note, that the last purification step, Gravity Flow Chromatography, has shown to be not necessary for truncated tyrosinase purity (hTyrC<sub>tr</sub>).</span></p>

[STEPS]
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TITLE: FCMPASS - Mac - Providing Read and Write Permission
AUTHORS: Joshua Welsh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">If you hear/receive error messages when opening FCMPASS or trying to create a database it likely due to you Mac not providing FCMPASS read and write permissions by default. This occurs most commonly on managed computers such as University/Government issued computers. This can be resolved with the following protocol.</div></div>

[STEPS]
?. Close FCMPASS before performing any of the following steps.
?. On the top menu bar click 'Go'.
?. Hold down the 'Option' key and 'Library' will appear on the menu.
?. Click 'Library'
?. In the finder window that pops up, click 'Application Support' and scroll down to 'Mathworks'.
?. Inside the 'Mathworks' folder, click 'MATLAB'.
?. Right click on the 'mcr_98' folder and click 'Get Info'
?. Unlock this folder by clicking on the padlock in the bottom right corner of the new window and enter your password. This will allow you to make changes to the folder.
?. Click on option to the right of your username and select 'Read & Write'.
?. Now click on the cog at the bottom and select 'Apply to enclosed items'.
?. You have now given the FCMPASS software permission to read and write cytometer databases. If this does not resolve issues you are having with the software please contact Joshua Welsh (joshua.welsh@nih.gov)
?. Click on 'Finder'
TITLE: 1M Potassium Citrate Buffer, pH 6.0
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
Potassium Citrate Buffer recipe

[STEPS]
1. 700 mL

2. 268.8 g tripotassium citrate or 293.5 g tripotassium citrate monohydrate (880 mM)
3. 26.3 g citric acid monohydrate (125 mM)
4. Adjust pH to 6.0 with 5 M KOH
5. Adjust to1000 mL

6. Autoclave
TITLE: Multi-electrode array (MEA)
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Multi-electrode array (MEA)

[STEPS]
1.  One day before plating, 6-well MEA plates (Axion Biosystems, M384-tMEA-6W) were coated with poly-D-lysine (Millipore Sigma P6407-5MG) in borate buffer
2. ,then washed six times with sterile water and aspirated prior use.
3. 150,000 cells in a 15 µl drop were plated on a 6-well MEA plate. Cultures were maintained for 17-21 days at 37 °C in 5% CO2
4. On the day of recording, MEA plates were equilibrated for 10 min on the pre-warmer reader at  37 °C in 5% CO2
5. 2 mins of firing activity was recorded at a 12.5 kHz sampling rate and filtered with a bandpass filter from 200 Hz to 2.5 kHz. Each MEA well contained 64 electrodes that detected changes in extracellular field potentials reflecting the spiking activity of neurons.Spike detection was performed with the adaptive threshold crossing algorithm with a threshold of 6× standard deviation using the AxIS software version 2.4 (RRID:SCR_016308) https://www.axionbiosystems.com/products/axis-software.  All spike trains were exported from AxIS software.
TITLE: Quantification of TH immunoreactivity
AUTHORS: Hong-Yuan Chu, Hong-yuan Chu

[DESCRIPTION]
This protocol describes quantification of TH immunoreactivity.

[STEPS]
SECTION: Procedure
1. TH signals from bilateral striatum and cortex are imaged under a 20x objective lens using identical imaging settings.
SECTION: Procedure
2. Open the confocal files in Image J software.
SECTION: Procedure
3. Using the “Rectangle” function to select an area (width x high = 150 x 150) in striatum sections from both hemispheres for TH quantification.
SECTION: Procedure
4. Using the “Measure” function to measure the gray value of the selected area and write down the mean value of striatum slice, Mstriatum-LHS and Mstriatum-RHS.
SECTION: Procedure
5. Using the “Rectangle” function to select an area (width x high = 150 x 150) in cortical sections from both hemispheres for TH quantification.
SECTION: Procedure
6. Using the “Measure” function to measure the gray value of the selected area and write down the mean value of cortical slice, Mcortex-LHS and Mcortex-RHS.
SECTION: Procedure
7. TH quantification =  .
TITLE: Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity
AUTHORS: Pilar Menendez-Gil, Carlos J. Caballero, Cristina Solano, Alejandro Toledo-Arana

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5' and 3' ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from </span><span style = "font-style:italic;">Staphylococcus aureus</span><span>. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.</span></div></div>

[STEPS]
TITLE: Immunocytochemistry (ICC) for Progenitor markers FOXA2 and OTX2
AUTHORS: Ning Xia

[DESCRIPTION]
This is the protocol for Immunocytochemistry (ICC) for Progenitor markers FOXA2 and OTX2.

[STEPS]
1. Grow the cells on either 8µm chamber slide or 48-well plate. (1x105cells/well)
-         Coating and plating densities can be found in the differentiation kit
2. Aspirate medium and rinse with 1x PBS
3. Fixation: Cover cells with warm 4% PFA and fix for 20 min at RT.
-         Cover the vessel and place in the hood
4. Rinse 3X in 1x PBS for 5 min each at the lab bench
5. Blocking and permeabilize for 30 min at RT with Blocking Buffer
6. 1º antibody incubation dilute in 1/3 blocking buffer + 1x PBS and incubate overnight in the cold room
-20º (box labelled antibodies for iPSCs)
-         FOXA2 Rb à (1:250)
-         OTX2 Mus IgG à (1:250)
o   Add 4µl of antibody per 1mL of prepared antibody solution
7. Rinse three times in 1x PBS for 5 min each
8. 2º antibody incubation for 1hr at RT in the dark
-         A11008 Alexa Flour 488 goat anti-rabbit IgG (HHH+L) 1:400 in PBS
-         A10036 Alexa Flour546 donkey anti-mouse IgG (H+L) 1:400 in PBS
o   Add 2.5µL of antibody per 1mL of prepared antibody solution  
*Combine these two antibodies in one tube. Ex. If you were making 1.6mL (for 8 wells) you would add 4µL of each antibody
9. Rinse 3X in 1X PBS for 5 min each
10. Incubate with DAPI at (1µg/mL) for 10min at RT (Stock is 1mg/mL which is 1000x)
a.      Add 1µl of Stock DAPI to 1mL of prepared solution.
11. 11.   Rinse 2X in 1X PBS and image.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Introduction</strong></p>
<p>In conventional MR examination glioblastomas multiforme (GBM), metastases and primary CNS lymphomas (PCNSLs) may show very similar appearance. The aim of the study was to evaluate usefulness of multiparametric DSC perfusion and diffusion MR imaging in the preoperative differentiation of these tumors.</p>
<p><strong>Material and Methods</strong></p>
<p>Fifty three solitary enhancing tumors (20 GBMs, 20 metastases, 13 PCNSLs) were enrolled in the study. Parameters of cerebral blood volume (rCBV), peak height (rPH), percentage of signal recovery (rPSR) and apparent diffusion coefficient (ADC) were assessed from the tumor core and peritumoral non-enhancing T2-hyperintense zone. </p>
<p><strong>Results </strong></p>
<p>Within the tumor core there were no differences in perfusion and diffusion parameters between GBMs and metastases. Compared to GBMs and metastases, PCNSLs showed significantly lower rCBV and rPH, ADC as well as higher rPSR values. Max rCBV with a cut-off value of 2.18 showed the highest accurracy of 0.98 in differentating PCNSLs from other tumors. To distinguish GBMs from metastases analysis of the peritumoral zone was performed showing significantly higher rCBV, rPH and lower ADC values in GBMs with the highest accuracy of 0.94 found for max rCBV at a cut-off value of 0.98.</p>
<p><strong>Conclusions </strong> </p>
<p>Max rCBV seems to be the most important parameter to differentiate GBMs, metastases and PSCNSLs. Analysis of max rCBV within a tumor core enables to distinguish hypoperfused (PCNSLs) from hyperperfused (GBMs and metastases) tumors while evaluation of max rCBV within the peritumoral zone is helpful to distinguish GBMs showing peritumoral infiltration from metastases surrounded by pure edema.</p>

[STEPS]
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TITLE: Implementation outcomes for agitation detection technologies in people with dementia: A systematic review protocol
AUTHORS: Lorna Smith, Nicolas Farina, Sube Banerjee

[DESCRIPTION]
Experiencing agitation can be particularly distressing for people living with dementia and their caregivers. Using technologies to automatically detect agitation can help monitor and intervene agitation occurrences. This systematic review aims to explore the early-stage implementation outcomes related to the use of agitation detection technologies, including video, wearables and multimodal. By adopting a taxonomy of implementation outcomes including acceptability, adoption, feasibility, fidelity, and implementation cost, this review seeks to provide insights crucial for real-world adoption of such technologies for people with dementia. Systematic searches will be conducted in the following databases; SCOPUS, PubMed, PsychINFO, IEEEXplore and CINAHL Plus. Expected results include qualitative themes and quantitative data related to these outcomes, obtained from the perspectives of the people with dementia, their caregivers, and organisational settings such as care homes. The review protocol outlines a comprehensive methodology for data synthesis, including critical appraisal and data extraction processes, to provide a nuanced understanding of the potential challenges and benefits associated with implementing agitation detection technologies in this population.

[STEPS]
1.
TITLE: Live-cell imaging: cell death assay
AUTHORS: gurvir.virdi

[DESCRIPTION]
Examining cell death in midbrain dopaminergic neurons using SYTOX Green

[STEPS]
1. Cell death is detected using SYTOX™ Green (SYTOX, Thermo Fisher Scientific) which is excluded from viable cells but exhibits green (SYTOX) fluorescence following a loss of membrane integrity. 

Hoechst 33342 (Hoechst, Thermo Fisher Scientific) is used as a nuclear cell stain.
2. Cells were incubated with 500 nM SYTOX and 10 uM Hoechst for 40 min at room temperature made up in HBSS.

3. Live-cell imaging is then performed using a confocal microscope.
3.1. Live-cell imaging is performed using a confocal microscope (Zeiss LSM 710 or 880 with an integrated META detection system). For confocal microscopes, illumination intensity is limited to 1% of laser output to prevent phototoxicity, and the pinhole is set to allow optical slice at approximately 1-2 μm. 
3.2. Hoechst is excited by 405 nm laser line with the emission between 405 nm to 470 nm.

SYTOX is excited by a 488 nm laser with emissions between 488 nm and 516 nm.
TITLE: Brimblecombe, K.R. et al. (2023) Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine, co-varies with risk factors for Parkinson’s. 
AUTHORS: Katherine Brimblecombe, Stephanie J Cragg

[DESCRIPTION]
This collection contains five protocols detailing methods used in Brimblecombe, K.R. et al. (2023) Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine, co-varies with risk factors for Parkinson’s. 

[STEPS]
TITLE: Y Choice test invertebrate
AUTHORS: Freddie-Jeanne Richard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Y  choice test designed for terrestrial invertebrase</div></div>

[STEPS]
?. [Device ]
Tests were performed using a Y-shaped choice chamber built in a plastic Petri dish (diameter: 8.7 cm; height: 1.2 cm; lid included) (Fig 2, from Beauché and Richard 2013 in Plos One). Rigid plastic tunnels were also used to create the device, and two plastic pipettes were sealed at the end of these tunnels to pulse air regularly into the system; the air was passed through sections (IIa) and (IIb) to spread the odor of the individuals.
?. [Animals placement and monitoring]
At the beginning of the experiment, the isopods were placed in the sections located in the three extreme parts of the tunnels:the two target females were placed in sections (IIa) and (IIb) (one in each section), and the tested male was placed in section (I). The male was then able to move into four other sections. The position of the target females in sections (IIa) and (IIb) was inverted between each replicate, and the target and tested individuals were used for only one test. The females were placed in their sections 15 minutes before the beginning of the test so that they could become accustomed to the new environment. The male was placed in section (I), andfrom the moment he entered the neutral section (NS), the time spent in the left section (LS) and the right section (RS) was monitored
TITLE: Bulk RNA Isolation (Ding)
AUTHORS: Reyka Jayasinghe, Feng Chen, Satok, Li Ding

[DESCRIPTION]
Bulk RNA Isolation - Ding Lab

[STEPS]
1. Add 1 ml Trizol to ~10 mg tissue.
2. Add 200 µL chloroform
3. Vortex for 1 min until homogenous.
4. Incubate at room temperature for 5 min
5. Spin samples in microcentrifuge at 12,000 rpm for 20 min. at 4°C
6. Transfer ~500 µL of clear (aqueous) top phase to a new 1.5 mL tube.
7. Add 500 µL of cold 2-propanol to the 1.5 mL tube.
8. Vortex for 30 sec. to mix 2-propanol/sample.
9. Put samples in –20°C for 3–24 hrs.
10. Spin samples in microcentrifuge at full speed for 20 min. at 4°C
11. Remove supernatant from tube.
12. Add 1 mL 80% Ethanol and vortex for 10 sec.
13. Spin samples in microcentrifuge at full speed for 5 min. at 4°C
14. Remove supernatant from tube.
15. Let tube sit at room temperature with top open for 5 min. to evaporate excess ethanol.
16. Add 87.5 µL of RNase-free water to the RNA pellet.
17. vortex for 5 min. to resuspend RNA, making sure that pellet is completely dissolved.
18. Spin in centrifuge briefly (Flash) to collect entire sample at the bottom of the tube.
19. Add 10 µL Buffer RDD and 2.5 µL DNase I (Qiagen)
20. Incubate on the benchtop (20–25°C) for 10 min.
21. Add 10 µL beta-mercaptoethanol per 1 mL buffer RLT
22. Add 350 µL Buffer RLT with beta-mercaptoethanol to the sample and mix well
23. Add 250 µL of 96–100% ethanol to the diluted RNA and mix well by pipetting.
24. Transfer the sample (700 µL) to an RNeasy MinElute spin column placed in a 2 mL collection tube, close the lid gently, and centrifuge for 15 sec. at 10,000 rpm at room temperature. Discard the flow-through.
25. Place the RNeasy MinElute spin column in a new 2 mL collection tube. Add 500 µL buffer RPE to the spin column. Close the lid gently, and centrifuge for 15 sec. at 10,000 rpm at room temperature. Discard the flow-through.
26. Add 500 µL of 80% ethanol to the RNeasy MinElute spin column. Close the lid gently, and centrifuge for 2 min. at 10,000 rpm at room temperature. Discard the flow-through and collection tube.
27. Place the RNeasy MinElute spin column in a new 2 mL collection tube. Open the lid of the spin column, and centrifuge at full speed for 5 min. at room temperature. Discard the flow-through and collection tube.
28. Place the RNeasy MinElute spin column in a new 1.5 mL collection tube.  Add 14 µL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge for 1 min. at full speed at room temperature.
TITLE: Mitochondrial Quality and Membrane Potential Detection
AUTHORS: mitsjoecohen  

[DESCRIPTION]
A large number of studies have shown that mitochondria are closely related to apoptosis, in which the decrease of mitochondrial transmembrane potential (MMP) is considered one of the earliest events in the cascade of apoptosis. It occurs before the appearance of the characteristics of nuclear apoptosis (chromatin condensation, DNA fragmentation). Once the mitochondrial transmembrane potential collapses, apoptosis is irreversible.

JC-1 has two states of monomer and polymer, and their emission spectra are different. In normal cells, the membrane potential of mitochondria is higher, and JC-1 aggregates in the matrix of mitochondria to form polymers, which can produce red fluorescence. In the early stage of apoptosis, the mitochondrial membrane potential decreases and JC-1 could not accumulate in the mitochondrial matrix. At this time, JC-1 is a monomer and could produce green fluorescence.

The decrease of cell membrane potential can be detected by the transformation of JC-1 from red fluorescence to green fluorescence, and the change of JC-1 fluorescence color can be used as an early detection index of apoptosis. The relative ratio of red and green fluorescence is often used to measure the proportion of mitochondrial depolarization.

[STEPS]
TITLE: E. coli Optical Quantification
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This procedure details how to culture </span><span style = "font-style:italic;">E. coli</span><span> K-12 in suspension and quantify cultured  bacteria using a spectrometer.</span></div></div>

[STEPS]
?. [Inoculate Culture]
Pipette BHI broth into 15 mL centrifuge tube.
10 ml
?. [Inoculate Culture]
Place tube in incubator or water bath to warm.
?. [Inoculate Culture]
Add lyophilized E. coli K-12 to warm BHI broth.
2 mg
?. [Inoculate Culture]
Close lid and shake gently.
?. [Inoculate Culture]
Incubate at for several hours.
?. [Quantify Concentration]
Dispense BHI broth into cuvette.
1 ml
This should be broth without bacteria and will serve as your reference for calculating absorbance.
?. [Quantify Concentration]
Carefully insert cuvette into spectrometer.
Wipe cuvette with delicate task wipes if necessary.
?. [Quantify Concentration]
Measure intensity at 600 nm.
?. [Quantify Concentration]
Repeat Steps 6-8 with the samples cultured for different durations.
Avoid pipetting any precipitation at the bottom of the culture tube. Draw only supernatant.
?. [Quantify Concentration]
Using the BHI broth without bacteria as I0, calculate the absorbance of each of the samples.
?. [Quantify Concentration]
Plot Absorbance as a function of time cultured. Include this plot, and the table of intensities and calculated absorbances in the 'Results' section of your lab report.
TITLE: Quality control for metagenomics data
AUTHORS: Qi Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Quality control for metagenomics data,including: remove low quality reads and host contamination reads.</div></div>

[STEPS]
?. Step1: remove low quality reads：We firstly calculate the accuracy probabilities of each base using the following equations:1)Q = -10 log10E2)P = 1 - EWhereQ is thePhred quality score of each base,Eis the error probability of each base.Then we calculate the overall accuracy probability(OA)of each readusing the following equation:For each read, an initial 30-mer seed sequence is selected at the 5’ end of the read and its overall accuracy, defined as OAseed, is calculated. To ensure high data quality, OAseedis defined as 0.9 usingm equal to 0with zero low quality bases allowed. Once the seed position of the read has been defined, the seed would extend to keep the longest contiguous read fragment in which the OA, defined as OAfragment, is above a defined accuracy threshold. In this study, weset OAfragmentequal to or greater than 0.8 usingmequal to 1.And the Perl scripts for overall accuracy based QC pipeline are freely available for download and reuse from  Github (https://github.com/Scelta/OAFilter).
?. Step2: remove host contamination reads by one command:'bowtie2 --very-sensitive -p $thread -x $host_bowtie2_index -1 $sample_r2 -2 $sample_r1 2> 02.rmhost/bowtie2.log | samtools view -h | samtools sort -n |samtools fastq -N -c 5 -f 12 -1 02.rmhost/$name.rmhost.1.fq.gz -2 02.rmhost/$name.rmhost.2.fq.gz'