eamag/protocols-with-steps huggingface dataset:
TITLE: Vandy - Modified Roux-en-Y Gastric Bypass in Mice
AUTHORS: Teri Stevenson, Vance L. Albaugh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This is the protocol for the modified gastric bypass in the mouse. In this procedure the stomach is bypassed and the food flows through the bypass arm directly into the jejunum. </div></div>

[STEPS]
?. Preoperative Care 1. All animals must be singly-housed, given Ensure 12 hours before surgery and have all bedding removed from the cages being used for housing. 2. Preoperative pain medications should be administered:     a. Ketoprofen (5 mg/kg)     b. Saline is given at the end of surgery and a second dose is given 24 hours later. 3. Ensure adequacy of anesthesia. 4. Place mouse on surgical board/surgical field over a warm water circulating blanket and immobilize gently. 5. Prep and drape animal sterilely.
?. Operation1. Begin the procedure by making a midline laparotomy incision with sharp surgical scissors. Be sure to stay on the linea alba and away from the rectus abdominus muscles. 2. Using cotton swabs, gently sweep the intestinal contents until the Ligament of Treitz is located. 3. Transect the intestine distal to the ligament and close the proximal end using 8-0 nylon. 4. A jejuno-jejunostomy is performed using a side to side anastomosis. 5. Ligate esophagus proximal to stomach using 6-0 silk. 6. An esophageal-jejunostomy is performed using 8-0 nylon. 7. Next, close the abdomen in a simple, two-layer, interrupted fashion. The first interrupted layer should be a simple, interrupted muscle layer with 6-0 monocryl suture. Using good technique suture the connective tissue and not the muscle proper. The second layer is a skin layer also done in a simple, interrupted fashion with 7-0 Prolene suture.
?. Postoperative Care 1. All mice receive 1.0 ml of warmed, sterile saline following the procedure before being placed in the recovery cages. 2. All mouse cages are kept partially on a veterinary-approved heating pad for 5 to 7 days postoperatively. 3. Mice will remain on an Ensure diet for 48 hours post-surgery with no bedding in the cage. 4. Mice are monitored until recovered from the procedure, which typically takes 7-14 days depending on the procedure. General behavior (i.e. bright/alert/responsive vs. depressed/obtunded) is monitored. 5. Pain medication is administered per protocol: ketoprofen 5 mg/kg post-op and again at 24 hours postoperative. 6. Additional pain medication may be needed depending on postoperative recovery.
TITLE: Efficacy of follow-up after intensive care unit (ICU) discharge for improving long-term outcomes and mental health in ICU patients and informal caregivers: a systematic review and meta-analysis protocol.
AUTHORS: Shodai Yoshihiro, Shunsuke Taito, Kota Yamauchi, Shunsuke Kina, Takero Terayama, Yusuke Tsutsumi, Yuki Kataoka, Takeshi Unoki

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;">Introduction</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Patients admitted in intensive care unit (ICU) and their informal caregivers acquired mental health disorder, which persisted after discharge. We describe a protocol for a systematic review for a question: Does follow-up after ICU discharge improve long-term outcomes and mental health compared with usual care in ICU patients and informal caregivers?</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;">Methods and analysis</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">We will follow the Preferred reporting items for systematic review and meta-analysis 2020 for conducting the research. We will search the literature on MEDLINE, the Cochrane Central Register of Controlled Trials, EMBASE, the Cumulative Index to Nursing and Allied Health Literature, and APA PsycInfo systematically.We will include randomized controlled trials that assessed the effect of follow-up after ICU discharge for patients and informal caregivers.Follow-up after ICU dischargewill beany service and program whichinclude psychological monitor, care and intervention. Primary outcomes for patients and informal caregivers were depression, post-traumatic stress disorder, and all adverse events. We will assess the certainty of evidence based on the Grading of recommendations, assessment, development and evaluation approach for each patient and informal caregiver.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;">Ethics and dissemination</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">As this research is a SR/MA of literatures, ethical approval is not necessary. We will publish the results of this SR/MA ina peer-reviewed journal.</div></div></div>

[STEPS]
TITLE: Raffaelea plant inoculation
AUTHORS: You Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to test pathogenicity of the ambrosia fungi Raffaelea.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Live, in vivo, imaging (GCaMP6F), Video Processing, and Analysis
AUTHORS: Bryan  Yoo, Jessica  Griffiths, Sarkis  Mazmanian

[DESCRIPTION]
Protocol for GCaMP6F imaging and analysis in gut neurons used in Yoo et al 2021

[STEPS]
SECTION: Preparation
1. AAV-PHP.S-CAG-GCaMP6F (1012VGs) was delivered systemically to WT C57.
SECTION: Preparation
2. 3-4 weeks after infection, mice were anesthetized with 2% isoflurane on a heating pad (Kent Scientific, Torrington, CT-DCT15) with plastic sleeve covers (Kent Scientific, Torrington, CT-DCT1520P). 
SECTION: Preparation
3. The abdominal cavity was surgically opened to expose the intestines. 
SECTION: Imaging
4. The proximal colon was identified, and this portion was placed on top of a stack of 4-6 glass microscopy slides (depending on size of animal) (VWR, Radnor PA-Cat. No. 48300-026). 
SECTION: Imaging
5. Tissue was secured onto glass slide with a biorthogonal silicon elastomer (Kwik-Sil) (World Precision Instruments, Sarasota, FL-KWIK-SIL), and a glass coverslip.
SECTION: Imaging
6.  Elastomer stiffens within 1 minute of application and coverslip must be held steadily to ensure a flat imaging surface. Anesthetized mouse is placed under an upright confocal microscope (Zeiss LSM 880). 
SECTION: Imaging
7. Using a 10X objective, GCaMP6F fluorescence was taken at 5Hz (1 image every 200ms). 
SECTION: Imaging
8. Periods of movement of tissue and luminal contents is normal during live imaging. 
SECTION: Processing &amp; Analysis
9. Cell tracking was performed in 2D by using TrackMate ImageJ plugin (https://imagej.net/TrackMate) and fluorescence intensity was recorded from cells. 
SECTION: Processing &amp; Analysis
10. Average background was determined by taking the fluorescence of a region of interest that did not contain a cell, over the duration of the video. Background was subtracted from cell fluorescence intensities.
TITLE: Mammalian Cell Culture: Refreshing Media
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains how to refresh the media of cultured mammalian cells grown in a tissue culture flask.</div></div>

[STEPS]
?. [Assessing Culture Health]
Assess cell health under light microscope before beginning.
?. [Refreshing media]
Using a serological pipet, remove old media from flask and transfer to waste beaker.
To avoid contamination, avoid touching the pipette tip to anything. Holding the flask such that the lid is pointed up will make it easier to remove all the liquid.
?. [Refreshing media]
Wash cells by pipetting warmed DPBS into the flask. [ for T-25 flask]
4 ml
1 ml
?. [Refreshing media]
Remove DPBS and dispose into waste beaker.
?. [Refreshing media]
Repeat the above two steps for a total of 2 washes.
?. [Refreshing media]
Pipette  -  warmed cell culture media into flask. [ for T-25 flask].
8 ml
12 ml
4 ml
?. [Incubate]
Spray flask generously with 70% ethanol solution before placing in CO2 incubator.
TITLE: High Performance Liquid Chromatography (HPLC) and sample preparation
AUTHORS: gurvir.virdi

[DESCRIPTION]
HPLC for metabolites in mDA neurons

[STEPS]
SECTION: Pre-sample prep
1. Prior to harvesting mDA neurons, all cells were incubated in phenol-free medium for 24 hours with and without the presence of 80 micromolar (µM) L-Dopa (Sigma).

SECTION: Extracellular metabolite sample prep
2. For extracellular metabolites, media after 24 hours was mixed 1:1 in 0.8 Molarity (M) ice cold perchloric acid.

SECTION: Extracellular metabolite sample prep
2.1. Samples were incubated on ice for 10 min , followed by centrifugation at 12000 x g, 5 min, 4 °C .

SECTION: Extracellular metabolite sample prep
2.2. The supernatant was removed and frozen in dry ice for HPLC analysis.
SECTION: Intracellular metabolite sample prep
3. For intracellular metabolites, after 24 hours, the cells were removed and pelleted by centrifugation at 1200 x g, 5 min, 4 °C.

SECTION: Intracellular metabolite sample prep
3.1. The cells were washed once in PBS and lysed on ice using lysis buffer (10mM Tris (pH 7.4), 1mM EDTA, 320mM sucrose in HPLC grade water). 
SECTION: Intracellular metabolite sample prep
3.2. Lysate was mixed with 1:1 in 0.8 Molarity (M) ice cold perchloric acid and incubated on ice for 10 min. 

SECTION: Intracellular metabolite sample prep
3.3. The samples were centrifuged at 12000 x g, 10 min, 4 °C and the supernatant was harvested and frozen for HPLC analysis.

SECTION: HPLC
4. Quantification of neurometabolites (DOPAC, 3-OMD, 5-HIAA, HVA and dopamine) was carried out using reverse phase HPLC and an electrochemical detector
SECTION: HPLC
4.1. The mobile phase (flow rate 1.5ml/min) contained 16% methanol, 20mM sodium acetate trihydrate, 12.5mM citric acid monohydrate, 3.35mM 1-octanesulfonic acid, 0.1mM EDTA disodium and adjusted to pH 3.45 with 12 M hydrochloric acid (HCl).
SECTION: HPLC
4.2. The stationary phase was maintained at 27 °C .

SECTION: HPLC
4.3. The detector electrode was set at 450mV and screening electrode at 50mV.
SECTION: HPLC
4.4. 50µl of sample was injected and calculated against a 500nM external standard solution of the 5 compounds of interest made in HPLC grade water acidified with 12M HCl.
SECTION: HPLC
4.5. Peak areas were quantified with EZChrom Elite™ chromatography data system software, version 3.1.7 (JASCO UK Ltd., Great Dunmow, UK).
TITLE: Cleaning colonial ascidians
AUTHORS: Simon Blanchoud, Marta  Wawrzyniak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol has been successfully used with Botrylloides diegensis and Botryllus schlosseri.</div></div>

[STEPS]
?. [Cleaning colonial ascidians]
Select the slides to be cleaned.
?. [Cleaning colonial ascidians]
Place them in the first container in a slide rack.
?. [Cleaning colonial ascidians]
Take one slide.
?. [Cleaning colonial ascidians]
Using the single-edge razor blade, wipe clean as much surface as possible.
?. [Cleaning colonial ascidians]
Make sure to avoid the colonies and the vascular system.
?. [Cleaning colonial ascidians]
Do not forget to clean the sides of the slides too.
?. [Cleaning colonial ascidians]
Using the wet paintbrush, wipe gently the systems and the tunic of the colonies.
?. [Cleaning colonial ascidians]
Try brushing	from the center of the colony towards its rim, to avoid detaching it.
?. [Cleaning colonial ascidians]
Brush out all the weird-looking tissue.
?. [Cleaning colonial ascidians]
Using the curved-edge scalpel blade, ablate the detached or decaying tissue.
?. [Cleaning colonial ascidians]
Use a rolling movement to avoid detaching more tissue: press the tip of the blade on the glass, do not slide it, roll the blade to press the edge through the tissue.
?. [Cleaning colonial ascidians]
Place the clean slide into the second container inside another slide rack.
?. [Cleaning colonial ascidians]
If the colony as grown too much or outside of the center of the slide, subclone it. Subcloning
?. [Cleaning colonial ascidians]
Optional: If slide needs to be super extra clean:
?. [Cleaning colonial ascidians]
Place the cleaned slide under the stereoscope.
?. [Cleaning colonial ascidians]
Inspect the slide.
?. [Cleaning colonial ascidians]
Use the wet paintbrush to clean the remaining dirt.
?. [Cleaning colonial ascidians]
Use the plastic tweezers to pluck recalcitrant pieces of foreign tissue.
?. [Cleaning colonial ascidians]
Let the slide rest in FASW for .
?. [Cleaning colonial ascidians]
Once all slides are clean, put them back in the system.
TITLE:     ELISA for quantification of IL-18 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Macrophages mostly make IL-18, which can be produced by hepatocytes and keratinocytes. Its principal target is a co-factor in Th1 cell induction. It causes interferon gamma production and enhances NK cell activity. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-18 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-18 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-18 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-18 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-18 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-18 standard dilutions and the human IL-18 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Splitting 96 Well Plates for gDNA Extraction and Continuing Culture
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Coat 96 well plate with Matrigel per well.
50 µl
?. Incubate at  for .
?. Prepare plate for expansion by aspirating Matrigel from plate.
?. Add  mTesR1 + 10 uM Rock Inhibitor to appropriate wells.
50 µl
?. Aspirate media from original plate.
?. Wash with  PBS and aspirate.
200 µl
?. Add  of 0.05% Trypsin.
25 µl
?. Incubate at for
?. Tap to lift cells from plate.
?. Check under microscope to ensure that cells have detached from plate.
?. Add  FBS and tap to mix.
50 µl
?. Transfer to 96 well PCR plate, while maintaining the location of each sample (this plate will be used for gDNA extraction).
50 µl
?. Transfer remaining cells (~30uL) to 96 well plate containing mTesR1.
?. Incubate at .
?. After , complete daily media changes with mTesR1.
TITLE: Online Survey Among Medical Professionals on Second Opinions in the Indication for Elective Caesarean Sections in Germany: Study Protocol
AUTHORS: Anke Kaulbert, Barbara Prediger

[DESCRIPTION]
Background: 
Caesarean section (CS) rates have been continuously rising worldwide over the last decade without being accompanied by significant health benefits. Various approaches are discussed internationally to limit the proportion of caesarean deliveries to the necessary cases. The proposed study aims to assess the medical professionals’ perspective, if second opinions should be used as a tool to reduce unnecessary CS. The goal is to examine criteria for or against obtaining a second medical opinion when an elective CS is indicated.

Methods: 
The study population will comprise established (prospective) gynecologists and midwives. The anonymous survey will be conducted online and the data will be evaluated using quantitative descriptive analysis.

Discussion: 
The survey is an opportunity to get a comprehensive picture from the professional perspective and paves the way for following research in this area.The results might be limited by a possible selection bias of the study population. 

Ethics: Ethical approval for the study was obtained by the Medical Ethics Committee of the University of Witten/Herdecke. Results will be published in a relevant scientific journal and communicated to interested participants (when demanded).
Ethical approval date: 15.07.2022 (S-127/2022)
Estimated start of the survey: September 2022.

Detailed information on our protocol can be found in the attached PDF file.

[STEPS]
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>There is the preparation of CTAB solution.</p>
<p> </p>
<p>CTAB Buffer</p>
<p>100ml 1M Tris HCl pH 8.0</p>
<p>280ml 5 M NaCl</p>
<p>40ml of 0.5 M EDTA </p>
<p>20 g of CTAB (cetyltrimethyl ammonium bromide) </p>
<p>Bring total volume to 1 L with ddH<sub>2</sub>O</p>
<p> </p>
<p>TE Buffer</p>
<p>10ml 1 M Tris HCl pH 8.0 </p>
<p>2ml 0.5 M EDTA</p>
<p>Bring total volume to 1 L with ddH<sub>2</sub>O</p>
<p> </p>
<p>1 M Tris HCl pH 8.0</p>
<p>121.1 g Tris Dissolve in about 700 ml of H<sub>2</sub>O.</p>
<p>Bring pH down to 8.0 by adding concentrated HCl (you’ll need about 50 ml).</p>
<p>Bring total volume to 1 L with ddH<sub>2</sub>O.</p>
<p> </p>
<p>0.5 M EDTA</p>
<p>186.12 g EDTA</p>
<p>Add about 700 ml H<sub>2</sub>O</p>
<p>16-18 g of NaOH pellets</p>
<p>Adjust pH to 8.0 by with a few more pellets, EDTA won’t dissolve until the pH is near 8.0</p>
<p>Bring total volume to 1 L with ddH<sub>2</sub>O.</p>
<p> </p>
<p>5 M NaCl</p>
<p>292.2 g of NaCl</p>
<p>700 ml H<sub>2</sub>O</p>
<p>Dissolve (don’t add NaCl all at once, it will never go into solution) and bring to 1 L</p>
<p> </p>
<p>7.5 M Ammonium acetate</p>
<p>57.81g ammonium acetate</p>
<p>~50 ml of H<sub>2</sub>O</p>
<p>Bring to 100 ml total volume</p>
<p> </p>
<p>Filter sterilize all stock solutions.To do this use a nalgene filter (stockroom catalog # PL-365 – $39.00 per case) with the faucet vacuum attachment located in the drawer below the waterbath across from the sink.</p>

[GUIDELINES]
<ul>
<li>Gloves must be worn at all times. Change gloves frequently and decontaminate often with DNA Away.</li>
<li>Use only unopened, sterile, aerosol-resistant pipette tips (filter tips) to minimize contamination of the pipette shaft and your samples. Clean the body of the pipettes regularly with DNA Away.</li>
<li>Discard pipette tips after each use to avoid cross-contamination. Eject pipette tips carefully to prevent aerosol formation or other splashing.</li>
<li>Be careful not to touch the inside lid of tubes as it will cross-contaminate samples.</li>
</ul>

[STEPS]
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TITLE: How to book a sorting slot in PPMS
AUTHORS: Daniel Gimenes

[DESCRIPTION]
Step by step protocol on how to book a cell sorter at EMBL's Flow Cytometry Core Facility

[STEPS]
SECTION: Access the PPMS calendar
1. Go to https://ppms.embl.de
SECTION: Access the PPMS calendar
2. Click on Flow Cytometry Core Facility

 

SECTION: Access the PPMS calendar
3. Login into your account

 

SECTION: Access the PPMS calendar
4. On the upper tabs, click on "BOOK"

 

SECTION: Access the PPMS calendar
5. Select one of the cell sorters that you want to book a slot

 

SECTION: Access the PPMS calendar
6. Use the options to move between weeks to find the desired date for your sorting

 


SECTION: Access the PPMS calendar
6.1. The calendar is colour coded:

 
Dark grey: Not available for booking
You will find this colour:
If you are not a novice (not a trained user), the over working and weekend hours slots will not be available for booking
If you are trying to book the instrument with less than 48 hours until the time of the appointment.

Light blue: Intervention (not available for booking)
This one highlights periods in which the instrument is blocked for scheduled interventions (such as a sorter bi-weekly aseptic cleaning) or for when the staff is not available to support the sorting (in such occasion, trained users might still have the opportunity to sort - please let us know).

*Notice that the instrument is blocked for the staff lunch period from 12:00-13:00 and it is also the period in which the staff has more time to change the nozzle between sessions.

Teal: Booked session that still has to be confirmed
Blue: Staff confirmed the session
Green: Staff confirmed the session for Operator Free (trained) users
Purple border: Session contains a comment to convey a special environment (such as 384 wp sorting)
Light grey: available slots for booking
SECTION: Access the PPMS calendar
6.3. A form will be shown for you to fill the information regarding your sorting. Once you are done, click on "Complete Booking"

 


SECTION: Access the PPMS calendar
6.2. To book a slot:

Select time slot by checking the 30 minutes segments boxes in the calendar
(Optional) If you are trained, check the "Operator Free" Box. If you want to add a comment to your booking check the "Comment" box.
Click on Book the selected sessions
 



SECTION: Access the PPMS calendar
6.4. Your newly booked slot should appear in the calendar!
The facility staff will then check and either confirm it directly, or if any issues arise you will be contacted for changes.