eamag/protocols-with-steps huggingface dataset:
TITLE:      ELISA for quantification of IL-25 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>Dendritic cells produced predominantly IL-25. It acts on various types of cells, including Th2 cells. It stimulates the synthesis of Th2 cytokine profile including IL-4 and IL-13. </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-25 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-25 present in the serum sample binds to antibodies adsorbed into 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-25 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-25 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) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-25 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-25 standard dilutions and the human IL-25 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Immunoblotting of I3 Neurons and dopaminergic neurons
AUTHORS: Nisha Mohd Rafiq, Pietro De Camilli

[DESCRIPTION]
This protocol describes the preparation of cell lysate from and iPSC-derived neurons (I3 Neurons, dopaminergic) and the immunoblotting procedure.

[STEPS]
SECTION: Cell culture and treatments
1. Grow I3 Neurons and dopaminergic (DA) neurons on six-well plates (3-5 × 105 cells/well). 
SECTION: Cell culture and treatments
2. After differentiation in their respective maturation media, wash the neurons with 1 mL ice-cold PBS.
SECTION: Cell culture and treatments
3. Lyse the cells  in 200 µL 1xRIPA lysis buffer (10X RIPA lysis buffer, Sigma-Aldrich) supplemented with complete™ EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche).
SECTION: Cell culture and treatments
4. Centrifuge the cells at 13000 x g for10 min. 
SECTION: Cell culture and treatments
5. Collect the supernatant and store at-20 °C.
SECTION: Cell culture and treatments
6. Determine the protein concentration in sample using Pierce BCA assay (ThermoFisher). 
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
7. Incubate appropriate volume of cell lysate solution at 95 °C for 5 min in SDS sample buffer containing 1% 2-mercaptoethanol (Sigma).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
8. Separate the extracted proteins by SDS-PAGE in Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) at 250V and transfer to nitrocellulose membranes (Bio-Rad) at 100 V for 60 min or 75 V for 120 min (for high molecular weight proteins: >150 kDa).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
9. Block the nitrocellulose membranes for 60 min with 5% non-fat milk (AmericanBIO) in TBST (tris-buffered saline [TBS] + 0.1% tween 20), then incubate   at 4 °C with primary antibodies.
 


SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
10. Wash the blots  with TBST, thrice, each 5 min.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
11. Incubate the blots with IRDye 680RD or 800CW (LI-COR) secondary antibodies (1:8000) for 60 min at Room temperature in TBST.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
12. Image blots using the Gel Doc imaging system (Bio-Rad) using manufacturer’s protocols.
TITLE: Protocol collection: Dissociation and FACS isolation of embryonic and post-embryonic C. elegans intestine cells for RNA-seq analysis
AUTHORS: Robert TP Williams, Erin Osborne Nishimura

[DESCRIPTION]
This is a collection of protocols for the isolation of C. elegans intestine cells through FACS from embryo, L1, and L3 stage . The protocols are focused on collecting material for RNA-seq analysis, but could be utilized for other biochemical assays. Troubleshooting suggestions are listed at key steps of the protocols.

To perform this experiment, perform the protocols in the following order:

1. Synchronized C. elegans culture on NGM plates for FACS isolation of intestine cells
2. [Embryo, L1, or L3] stage C. elegans  dissociation for FACS isolation and RNA-seq analysis of intestine-specific cells
3. FACS isolation of intestine-specific C. elegans cells

A single experiment cycle takes approximately two weeks to complete.

[STEPS]
TITLE: Intracardiac perfusion with fixative for anatomical studies
AUTHORS: Janet Keast, Peregrine Osborne, Nicole Wiedmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is suitable for preserving tissues for anatomical studies of organs, ganglia, spinal cord or brain in adult rats. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation.</div></div>

[STEPS]
?. [Preparation for perfusion]
Make up the following solutions:Perfusion prewash solution: To 300 ml 0.9% sodium chloride (w/v) add 3.75 ml 1% sodium nitrite (w/v) and 0.11 ml heparin (5000 IU/ml). This is made up immediately prior to use.Perfusion fixative: 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. This is made up no longer than 48h prior to use, stored at 4C and brought to room temperature on the day of perfusion.
?. [Perfusion]
Induce anesthesia by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).
?. [Perfusion]
After opening the chest cavity, inject the left ventricle with mixture of 0.25 ml heparin (5000 IU/ml) and 0.5 ml 1% sodium nitrite.
?. [Perfusion]
A needle connected to tubing (T-connector to prewash and perfusion solutions), and a peristaltic pump (setting: 50 ml/min) is then inserted into the left ventricle and clamped into place using hemostats. Make a small incision in the right atrium to drain blood and perfusate during the procedure.
?. [Perfusion]
Perfuse with pre-wash solution until the fluid flowing from the right atrium is clear, the liver and extremities are pale (typically 2-3 min).
?. [Perfusion]
Perfuse with fixative for a minimum of 10 minutes, by which time the organs have stiffened and the neural tissues will be well preserved.
?. [Perfusion]
Dissect tissues required for analysis and place in fixative for a further 1h at 4C.
?. [Post-perfusion]
Wash tissues with phosphate-buffered saline (PBS; 0.1 M, pH7.2), 3 x 30 min washes.
?. [Post-perfusion]
Store in PBS containing 0.1% sodium azide at 4C until used for immunohistochemistry or other microscopic analysis.
TITLE: MGH Harvard SenNet Processing murine lung for paired single cell RNA-seq and mass spec
AUTHORS: gshipkovenska

[DESCRIPTION]
Protocol for obtaining single cell suspension of murine lung.

[STEPS]
SECTION: Dissection
1. Euthanize mice by CO2 method.
SECTION: Dissection
2. Perfuse lungs via right ventricle.
SECTION: Dissection
3. Dissect out trachea and lungs and place in HBSS on ice.
SECTION: Single cell suspension
4. 
SECTION: Count cells with heamocytometer
14. Count two samples each per lung and note down the exact number of cells. This information is important for scRNA library prep. We routinely recover 8-10 million cells / mouse with this protocol.
SECTION: Single cell suspension
5. Pipet 1ml digestion solution on the lid of a 10cm dish. Place the lung in it. Using a razor blade, "mince" the lung, holding one tip with the forceps.
SECTION: Single cell suspension
6. Pipet the sample into 4ml of the digestion solution / lung (8ml per mouse) and incubate at 37*C for 30min with rocking (in a 15ml falcon tube).
SECTION: Single cell suspension
7. Use a Pasteur pipet to triturate suspension until tissue is fully dissociated.
SECTION: Single cell suspension
8. Pass the suspension through a 100 μm cell strainer into 50-mL tubes and use 10 mL of DPBS to pass through the strainer to wash it.
SECTION: Single cell suspension
9. Centrifuge at 1000 × g for 5 min at 4°C, discard the supernatant.
SECTION: Single cell suspension
10. Add 1-2 mL of 1× RBC lysis buffer to resuspend the pellet and incubate for 5 min at 20°C–26°C.
SECTION: Single cell suspension
11. Add 10 mL of DPBS to stop the lysing,
SECTION: Single cell suspension
12. Centrifuge at 1000 × g for 5 min at 4°C, discard the supernatant.
SECTION: Single cell suspension
13. Resuspend with 200 uL of 0.5% BSA in PBS. Keep on ice.
SECTION: Prepare samples for scRNA-seq and scMS
15. Split lung samples into 100,000 cell aliquots, according to the cell counts determined above.
SECTION: Prepare samples for scRNA-seq and scMS
16. Keep one aliquot on ice for the scRNA-seq sample.
SECTION: Prepare samples for scRNA-seq and scMS
17. For the remaining aliquots, spin down at 1000 × g for 5 min, discard the supernatant and resuspend in 90% FBS, 10% DMSO and freeze at -80*C. These are the scMS samples.
SECTION: scRNA library preparation
18. Prepare according to manufacturer's protocol:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is part of the VERVE holiday drive to collect exotic off-the-shelf laboratory recipes. More details <a href="https://www.protocols.io/g/verve-net/news/call-for-recipes" target="_blank">here</a>.<br /><br />Timing is everything when preparing a large meal.  Unfortunately, for the truly epic holiday meals – like Thanksgiving – there never seems to be enough oven space to ensure everything arrives at the table steaming.  Something has always cooled, collapsed, or coagulated before the turkey is ready to carve.<br />This is where the autoclave comes in:  It’s built to hold loads at around 250 degrees Fahrenheit, and since it subjects them to twice normal atmospheric pressure, it tends to cut cooking times in half. Autoclave cook-time for a small to mid-sized turkey should be less than 2 hours.<br />Wanna impress the in-laws this year?  Bring home your favorite tabletop food-grade autoclave (not your Priorclave, though; those are <em>research</em>-grade autoclaves!) and put it to use on the big day.<br /><br /><br />

[GUIDELINES]
The tips in this protocol are adapted from advice found in the <a href="http://www.meltedmint.com/forum/viewtopic.php?f=7&amp;t=34" target="_blank">MeltedMint.com</a> forums and conversations with Priorclave staff.

[STEPS]
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?. 
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?. 
?. 
?. 
?. 
?.
TITLE: An Overview of Host Cell Protein
AUTHORS: 181830691 , hy

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Host cell proteins are process-related impurities that are expressed by host cells used to produce biologic drug proteins. During the purification process, most HCP is removed, but residual HCP amounts remain in distributed products such as monoclonal antibodies, antibody-drug conjugates, therapeutic proteins, vaccines, and other protein-based biopharmaceuticals.</div></div>

[STEPS]
TITLE: Protein expression in OnePot PURE cell-free system
AUTHORS: Konstantinos Ragios

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol we explain the procedure of protein expression in the OnePot PURE cell-free system.</div></div>

[STEPS]
?. [Protein expression]
For a 5μl reaction add to a tube 2μl of Energy solution, 0.65μl of Protein solution, 0.9μl of Ribosome solution, the DNA template (5nM final concentration in the reaction) and if needed add water to reach the final volume.
The minimum reaction volume is 5μl while the suggested one in 10μl. For any final reaction volume (e.g. Χ μl) you just need to multiply the volumes of the components needed by a factor of X/5.
If you haven't produce ribosomes then any store-bought product can be used.
?. [Protein expression]
Centrifuge for a few seconds and then your solution will start reacting.
?. [Protein expression]
If you want to measure it with a plate reader, ideally you should add each component in different corners of the well, so the reaction will not initiate before adding the plate on the reader. The centrifuge for a few seconds.
TITLE: Conditions for growth of Rhodobacter sphaeroides in different media 
AUTHORS: Jaya  K Yakha, Deborah K Hanson, Rosemarie Wilton, laible

[DESCRIPTION]
This protocol provides a method for culturing Rhodobacter sphaeroides wildtype 2.4.1 and its KO strains in shake flask and BioTek Epoch 2, 48-well microplates.  A baffled side arm flask is used but other flasks can be used with appropriate modifications.  

[STEPS]
SECTION: Conditions for growth of Rhodobacter sphaeroides in GYCC medium in shake flasks
1. Inoculate a GYCC agar plate from a glycerol stock containing appropriate antibiotic (if necessary) and incubate at 33 °C in an incubator in dark. 


SECTION: Conditions for growth of Rhodobacter sphaeroides in GYCC medium in shake flasks
2. After 4320 min pick one colony from the plate and inoculate 80 ml fresh GYCC medium with appropriate antibiotic (if necessary) in 125 mL baffled side arm flask. 



SECTION: Conditions for growth of Rhodobacter sphaeroides in GYCC medium in shake flasks
3. Incubate at 33 °C shaking at   for 72 -96 hours.


SECTION: Conditions for growth of Rhodobacter sphaeroides in GYCC medium in shake flasks
4. Monitor the growth of the culture with regular measurement of turbidity. Side-arm flasks are especially convenient for the determination of culture turbidity with a Klett-Summerson colorimeter. The equivalent OD600 may also be used if side arm flasks are not used.

SECTION: Conditions for growth of R. sphaeroides in GYCC or MR26 medium in Microplate
5. Inoculate GYCC or MR26 agar plates from a glycerol stock containing appropriate antibiotic if necessary and incubate at 33 °C . 

SECTION: Conditions for growth of R. sphaeroides in GYCC or MR26 medium in Microplate
6. After 4320 mins pick one colony from the plate and inoculate 25 ml fresh medium of GYCC or MR26 containing appropriate antibiotic (if necessary) in 50 mL flask


SECTION: Conditions for growth of R. sphaeroides in GYCC or MR26 medium in Microplate
7. Incubate at 33 °C with shaking at   for 72 -96 hours.

SECTION: Conditions for growth of R. sphaeroides in GYCC or MR26 medium in Microplate
8. Monitor the growth of the culture with measurement of turbidity. Once the culture reaches to the log phase (OD600~0.7), the starter culture is ready for inoculation of the microplate.

SECTION: Conditions for growth of R. sphaeroides in GYCC or MR26 medium in Microplate
9. For each well 600, µl of fresh GYCC or MR26 medium was added. It is recommended to use Microplate Greiner BioOne 48 well (Item no. 677180) for compatibility with a BioTek Epoch2 microplate reader
SECTION: Procedure Details in microplate reader
9.1. Plate Type: Greiner BioOne 48-wellv2 (Use plate lid)
Eject plate on completion
Set Temperature: Setpoint 33°C, Gradient 2 °C
Start Kinetic: Runtime 96:00:00 (HH:MM:SS), Interval 0:15:00, 385 Reads 
Shake Double Orbital: Continuous
Frequency: 548 cpm (2 mm)
Read:A600
Absorbance Endpoint
Full Plate
Wavelengths:600
Read Speed: Normal, Delay: 200 msec, Measurements/Data Point: 8 
End Kinetic
10. Once the run is complete, export the data in a .txt or .xls file and save in an appropriate location. Analyze and visualize the data by any software.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">This protocol lyses symbiodinium cells with heat and pellets debris, producing material suitable as crude DNA template for robust PCRs. It is convenient for rapid strain identification from liquid cultures and from </span><em><span style="font-weight: 400;">Symbiodinium</span></em><span style="font-weight: 400;">-hosting Aiptasia</span><em><span style="font-weight: 400;"> </span></em><span style="font-weight: 400;">anemones.</span></p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Vandy - Hyperinsulinemic-Hypoglycemic clamp
AUTHORS: Li Kang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Mice with catheters implanted in the jugular vein (infusions) and carotid artery (sampling) are used for this procedure (V3002). The hyperinsulinemic hypoglycemic clamp involves a constant rate insulin infusion with a fall in blood glucose that is controlled by feed back from regular glucose measurements. Blood glucose is then clamped at a hypoglycemic level. The hypoglycemic clamp is used to test hypoglycemic counterregulation and the functionality of the hypothalamic-pituitary-adrenal axis.</div></div>

[STEPS]
?. Surgical catheterization of the carotid artery and jugular vein in mice at least 5 days prior to the day of the study (refer to protocol for Surgical Catheterization of the Carotid Artery and Jugular Vein).
?. Weigh mouse and start fast (suggested starting time between 7:00 and 8:00 AM) by placing mouse in a plastic container with fresh bedding.
?. Mouse is hooked up to the swivel 3 hours into fasting (refer to protocol for Hyperinsulinemic-Euglycemic Clamp for detailed set-up and connections).
?. After a total of 5 hr fast, a constant infusion of insulin starts and glucose is monitored every 10 min and is allowed to fall and is fixed at a hypoglycemic level (~60mg/dL) by adjusting the infusion of 20% glucose.
?. Donor blood is infused to jugular vein catheter throughout the study to prevent a fall of hematocrit.
?. Catecholamines and glucagon levels are measured at 0, 30, and 120 min. Insulin is measured at 0 and 120 min.
?. At the end of the study, mouse is anesthetized and tissues of interest are harvested and frozen in liquid nitrogen.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Multicriteria desicion analysis (MCDA) is a tool used to perform quantitative benefit-risks assessment (BRA) anlysis in drugs, procedures and devices in health. It allows comparisons in the same indication, that allows quantitative BRA balances, is useful to shows results in a transparency and consistency manner and also to communicate easily to the stakeholders.</p>
<p> </p>
<p>MCDA has emerged as a promising tool in the decision context for drugs, although its use has been relegated to assess drugs in the context of medicine reimbursment and a few for benefits, risks and others assessments in medicines for a particular indication. One of the major advantages of MCDA is the inclusion of criterian weights that is "preferences". this roadmap to formulate a MCDA compile the recomendations of IMI-PROTECT and ISPOR task force to easy, explicit and transparent election of criteria included in a MCDA.</p>

[GUIDELINES]
<p>IMI-PROTECT Benefit-Risk Group RECOMMENDATIONS REPORT; Recommendations for the methodology and visualisation techniques to be used in the assessment of benefit and risk of medicines</p>
<p>Multiple Criteria Decision Analysis for Health Care Decision Making—An Introduction: Report 1 of the ISPOR MCDA Emerging Good Practices Task Force</p>
<p>Multiple Criteria Decision Analysis for Health Care Decision Making—Emerging Good Practices: Report 2 of the ISPORMCDA Emerging Good Practices Task Force</p>

[STEPS]
?. 
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?. 
?. 
?.
TITLE: Socio-demographic and lifestyle influences on falls among the elderly persons living in Gatanga Sub-County, Murang’a County, Central Kenya: A cross-section study
AUTHORS: Ernest Kimani, Simon Karanja, Lawrence Muthami

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">An estimated one-third of the elderly people worldwide sustain falls every year, which contributes to over 20% of injuries in this age group. In the central region of (Mount) Kenya, despite increasing awareness about this condition, the magnitude as well as the socio-demographic and lifestyle factors leading to falls in the elderly are not well known. </div><div class = "text-block">The main objective of this study was to investigate the prevalence of falls, their outcomes, and the factors influencing their occurrence among the elderly persons living in Gatanga Sub-County, Murang’a County, central Kenya. An analytical cross-sectional study design was used. Using systematic random sampling, 426 out of 9247 study participants were identified and interviewed at home. Using a structured interview form, data was collected on socio-demographic, socio-economic, lifestyle, and medical factors, as well as on fall or non-fall status from September 2016 to August 2017.</div><div class = "text-block">With 41%, the prevalence of falls amongst the elderly in this region is high. The farming lifestyle involving animal husbandry and medical illnesses are significantly associated with falls. To reduce the occurrence of falls, we need to better understand the underlying contributing factors and the integration of relevant preventive measures that reduce the risk of falls in communities of the elderly.</div></div>

[STEPS]