eamag/protocols-with-steps huggingface dataset:
TITLE: Plasma/serum sample preparation for untargeted MS-based metabolomic
AUTHORS: Carole MIGNÉ, charlotte JOLY, Stéphanie DURAND, Estelle Pujos-Guillot

[DESCRIPTION]
The following protocol describes the preparation of serum and plasma samples for non-targeted analysis by mass spectrometry coupled to liquid or gas chromatography, as proposed by the PFEM (MetaboHUB-Clermont).

[STEPS]
SECTION: Sample preparation
1. Thaw the samples according to these conditions depending on the analysis to be carried out :

 


SECTION: Sample preparation
5. Extract protein by adding  ice-cold methanol to each sample under the following conditions :  


 

SECTION: Sample preparation
6. Mix and placed the samples at -20 °C for 30 min.
    

SECTION: Sample preparation
7. Centrifuge samples 15493 x g, 10 min, 4 °C (Sigma 3-16PK, Fischer Bioblock Scientific).

The supernatant is divided into three aliquots. A intermediate pooled QC sample is prepared by mixing 10 µLfrom each extracted samples (per day or per batch).

 


SECTION: Sample preparation
9. Store samples for LCMS analysis at -80 °C until further analysis

SECTION: Sample preparation
8. Dry samples completely using a EZ2.3 genevac system (Biopharma Technologies France). 

 
    The following program is used:  
 
Programme HPLCFinal Stage 10 minReduce Odour OffLamp Off
 
 

 
SECTION: Sample preparation
10. Derivatisation for GCMS analysis :
SECTION: Sample preparation
10.4.    
Add 80 µLof BSTFA (1%TMCS) in the mixture and derivatize at 70 °C for 60 min 

SECTION: Sample preparation
10.2. Dissolve the dry samples in adding 80 µL of methoxylamine solution to each vial.  

SECTION: Sample preparation
10.3. Vortex vigorously for 1 min and incube at 37 °C for 1440 min (in order to inhibit the cyclization of reducing sugars and the decarboxylation of α-keto acids).

SECTION: Sample preparation
10.5. Waiting a few minutes and transfer 50 µL of derivatized mixture in a glass vial containing 100 µL of heptane prior to injection. 
Store blank and biological samples at 4 °C until further analysis
SECTION: Sample preparation
3. Prepare blank sample for GCMS analysis : add 100 µL of   in Eppendorf tube

SECTION: Sample preparation
2.  Add 100 µL of serum or plasma in Eppendorf tubes.  

SECTION: Sample preparation
4. Prepare 2 mL of an internal standard solution [13C1]-L-valine (0.2 mg/mL  ) in   , store at -20 °C for GCMS analysis

SECTION: Sample preparation
10.1. Prepare a fresh methoxylamine solution (15 mg/mL) in pyridine. The volume of this solution depends on the number of samples to be analysed (80 µL for each samples)

SECTION: Sample preparation
10.6. As well as a sample pool is formed from 10 µL of each extracted and derivatized sample to monitor the drift of the spectrometer during GCMS analysis.
Transfer 50 µL of sample pool in a glass vial containing 100 µL of heptane prior to injection, repeat this step 8 times (preprocessing analysis)
Store pool samples at 4 °C until further analysis
TITLE: Fluorescence intensity analyses
AUTHORS: Ayse Ulusoy, Sinead O'Sullivan, Michael Helwig, Angela Rollar, Shirley Lee, Michael Klinkenberg, Rita Pinto-Costa, Donato Di Monte

[DESCRIPTION]
Fluorescence intensity analyses

[STEPS]
1. 
Collect confocal z-stack images from fluorescent-labeled tissue samples. Here we used DHE-treated mice tissue counter-stained with human alpha-synuclein or tissue co-immunolabeled with a human alpha-synuclein and SynO2 antibodies.  
SECTION: Ox-DHE fluorescent signal measurement
2. Create a 3D surface rendering model of h-alpha-synuclein–immunoreactive
DMnX neurons using the Imaris software. 
SECTION: Ox-DHE fluorescent signal measurement
3. By applying a constant intensity threshold select ox-DHE puncta and filter through h-alpha-synuclein–immunoreactive neuronal surfaces. This will allow for specific detection of ox-DHE puncta within immunoreactive
neurons
SECTION: Ox-DHE fluorescent signal measurement
4.  Quantify puncta on a per-cell basis using the quantification tools. 
SECTION: Syn-O2 fluorescence intensity measurement
5.  Generate 2D images that from 5 µm-thick z-stack images using maximum
intensity projection function of the Zen software (Carl Zeiss). 
SECTION: Syn-O2 fluorescence intensity measurement
6. Select human alpha-synuclein labeled neurons by applying a 120µm2
size exclusion filter 

SECTION: Syn-O2 fluorescence intensity measurement
7. Measure Syn-O2 intensity within these cells using the measure tool.
TITLE: Starvation & washing protocol for anaerobic ciliates
AUTHORS: Roxanne Beinart, Johana Rotterova

[STEPS]
?. [Cell picking]
To pick ciliate cells, use a sterile drawn glass Pasteur pipette, a sterile depression slide with multiple wells, and inverted microscope. Transfer several hundred live cells into clean media (0.22 μm filtered).
?. [Cell washing]
Then, pass the cells through 3 wells of 0.22 μm filtered clean media.
?. [Cell washing]
Finally, pass the remaining healthy looking and active cells into a sterile 1.5 ml eppendorf tube with 0.5 ml of clean, 0.22 μm filtered and hypoxic media.
?. [Starvation]
Let the cells starve for 4 - 12 hours, if the species or group allows this.
?. [Starvation]
As soon as you determine absence of food vacuoles in sufficient amount of ciliate cells and at the same time, no bacterial growth is present in the media, end the starvation and proceed to wash.
?. [Starvation]
Using clean glass micropipette, isolate one cell after each hour, place it onto a microscope slide and observe under a light microscope. Depending on the conditions of the cell, presence of full food vacuoles and overall health, extend or decrease the starvation time.
?. [Starvation]
Starvation time has to be specifically adapted to each ciliate taxa, depending on oxygen tolerance, general endurance, and sensitivity to other conditions.
?. [Starvation]
If any bacterial growth appears during starvation, transfer the cells into clean filtered media, proceed to wash three times again, and place cells into a new eppendorf tube with clean filtered hypoxic media.
?. [Washing]
Carefully wash starved cells in a drop of sterile hypoxic filtered media.
?. [Washing]
Transfer cells through four to six drops of sterile hypoxic filtered media, depending on the cell conditions, reduce number of washings if the cells tend to burst and increase if you see free-living prokaryotes.
?. [Washing]
Transfer 50 - 100 cleaned starved cells into a sterile eppendorf tube with 200 μl of DNA Shield product (Zymo Research) and proceed to DNA isolation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Preparation of 1.5 mg/mL Sera-mag magnetic particles for DNA purification, a low cost Agencourt Ampure XP reagent substitute</p>
<p> </p>
<p>Adapted from B. Faircloth &amp; T. Glenn, 11.19.11, Ecology and Evolutionary Biology, UCLA</p>
<p><br />https://ethanomics.files.wordpress.com/2012/08/serapure_v2-2.pdf</p>

[BEFORE_START]
<p>Prepare 1x TE, filter sterilize (0.2 µM).</p>

[GUIDELINES]
<p>Materials:</p>
<p> </p>
<table width="658">
<tbody>
<tr>
<td width="169">Component</td>
<td width="99">Vendor</td>
<td width="99">part #</td>
<td width="48">amt/mL</td>
<td width="38"> </td>
<td width="71">in 50 mL final</td>
<td width="65">final [ ]</td>
<td width="33">500</td>
<td width="36"> </td>
</tr>
<tr>
<td width="169">Sera-mag SpeedBeads carboxylate modified magnetic particles, 5% solids, 50 mg/mL (Hydrophobic)</td>
<td width="99">GE Healthcare Life Sciences</td>
<td>44152105050350</td>
<td>0.030</td>
<td>1.5</td>
<td>1.5 mL/75 mg</td>
<td>1.5 mg/mL</td>
<td>15</td>
<td>mL</td>
</tr>
<tr>
<td>PEG-8000</td>
<td>Amresco</td>
<td>159</td>
<td>0.22</td>
<td>11.0</td>
<td>11 g</td>
<td>22% w/v</td>
<td>110</td>
<td>g</td>
</tr>
<tr>
<td>NaCl, 5 M</td>
<td> </td>
<td> </td>
<td>0.2</td>
<td>10.0</td>
<td>10 mL</td>
<td>1.0 M</td>
<td>100</td>
<td>mL</td>
</tr>
<tr>
<td>TE, 100X</td>
<td>Sigma</td>
<td>T9285</td>
<td>0.01</td>
<td>0.5</td>
<td>500 µL</td>
<td>1 X</td>
<td>5</td>
<td>mL</td>
</tr>
<tr>
<td>Tween 20</td>
<td>Sigma</td>
<td>P9416</td>
<td>0.55</td>
<td>27.5</td>
<td>27.5 µL</td>
<td>0.055 % v/v</td>
<td>275</td>
<td>µL</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p>50 bp ladder</p>
<p>1x (10 mM) Tris-HCl (pH 8.0 - 8.5)</p>
<p> </p>
<p>Note:  Sera-mag non-SpeedBeads and/or hydrophilic types can be used.  If non-SpeedBeads are used, more</p>
<p>time will be needed during magnetic separations.</p>
<p> </p>
<p>Equipment:</p>
<p> </p>
<p>Magnetic stand for 1.5 mL Eppendorf tubes</p>
<p>Magnetic stir plate</p>
<p>Stir bars</p>
<p>Laboratory scale</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Microplate reader operating procedure V.2
AUTHORS: Zhujun Wei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We use Tecan Spark® multimode microplate reader to measure absorbance.</div></div>

[STEPS]
?. 10mTurn on the power and warm up for 10 minutes.
?. Open the program and select "New" to create a new process.
?. Select "Absorbance" in the left column. There is a schematic of a 96-well plate in the middle of the screen.
?. You can select the hole to be used according to the spotting hole, and the selected hole will turn blue.
?. Put in the 96-well plate, set the parameters, click "start" to measure the absorbance, and then remove the 96-well plate.
?. Wait a moment, it will generate an Excel file with results and various parameters. Save and copy the file to the u disk and process the data.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The human umbilical vein endothelial cell line EA.hy926 (ATCC; Mannasas, VA, USA) was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) in an atmosphere of 5% CO2/95% air and 37°C.</p>
<p>EA.hy926 cells (10<sup>5</sup>cells/plate) were seeded in transwell inserts (Corning® Biocoat<sup>TM</sup> Cell Culture Inserts Collagen, Type I Rat Tail, 24-Well, 3 µm; Corning, NY, USA) and were cultured to confluence at 24 h. The cells were starved overnight and then activated with LPS (1 µg/ml) for 4 h. Upper chamber media, containing LPS (1 µg/ml) and soluble endoglin (500 ng/ml) for their respective treatments, were replaced with FITC-Dextran (40 kDa) at 1 mg/ml in DMEM. The bottom chambers were also replaced with DMEM. After 24 h at 37°C the inserts were removed, and the amount of fluorescence in the bottom chambers was measured using a fluorescence plate reader (Fluoroskan Ascent FL; Thermo Electron Corporation, Waltham, MA, USA).</p>

[GUIDELINES]
<p>Avoid repeated freeze-thaw cycles</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol for the making of 1M Propionic Acid for the use in Drosophila food. </p>

[BEFORE_START]
<p>Be sure to have the following materials: </p>
<ul>
<li>Propionic Acid, Sodium Salt</li>
<li>Hydrochloric Acid</li>
<li>Distilled water</li>
<li>Deionized water</li>
</ul>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Protocols for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells
AUTHORS: Marco A. de Oliveira, Lilian H. Florentino, Thais T. Sales, Rayane N. Lima, Luciana R. C. Barros, Cintia G. Limia, Mariana S. M. Almeida, Maria L. Robledo, Leila M. G. Barros, Eduardo O. Melo, Daniela M. Bittencourt, Stevens K. Rehen, Martín H. Bonamino, Elibio Rech

[DESCRIPTION]
This is a collection of protocols for assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells in human, animal and plants. Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome of a host. Their ability to rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as genetic editing tools with high host plasticity. Despite advances in their use in prokaryotic cells, only a few Ints are currently used as gene editors in eukaryotes, partly due to the functional loss and cytotoxicity presented by some candidates in more complex organisms. To help expand the number of Ints available for the assembly of more complex multifunctional circuits in eukaryotic cells, this protocol describes a platform for the assembly and functional screening of serine-integrase-based genetic switches designed to control gene expression by directional inversions of DNA sequence orientation. The system consists of two sets of plasmids, an effector module and a reporter module, both sets assembled with regulatory components (as promoter and terminator regions) appropriate for expression in mammals, including humans, and plants. The complete method involves plasmid design, DNA delivery, testing and both molecular and phenotypical assessment of results. This platform presents a suitable workflow for the identification and functional validation of new tools for the genetic regulation and reprogramming of organisms with importance in different fields, from medical applications to crop enhancement, as shown by the initial results obtained. This protocol can be completed in 4 weeks for mammalian cells or up to 8 weeks for plant cells, considering cell culture or plant growth time.

[STEPS]
TITLE: Protein interaction analysis of KaiC3 with various Kai homologs via yeast two-hybrid experiments (Growth Assay)
AUTHORS: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to investigate protein-protein interaction via yeast two-hybrid experiments. It describes the yeast-two hybrid method relying on the activity of the histidine-synthetase. If the proteins interact, the yeast cells are able to grow on selective medium without histidine. </div></div>

[STEPS]
?. [Preparation of buffers and media]
PrepareCSM ‑Leu ‑Trp agar (complete supplement mixture lacking leucine and tryptophan) CSM ‑Leu ‑Trp -His agar (complete supplement mixture lacking leucine, tryptophan and histidine) 3-AT solution, 1M
?. [Transformation of yeast cells]
Perform the transformation of yeast cells according to manufacturer's guidelines using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and select transformed cells on complete supplement mixture lacking leucine and tryptophan (CSM ‑Leu ‑Trp) at 30 °C for 3–4 days.
?. [Restreaking of colonies]
Prepare plates: CSM -Leu -Trp -His + 3-AT (interaction plate)Determine the appropriate concentration of 3-AT by adding different concentrations of 3-AT to the interaction plate   -  Possible concentrations of 3-AT: 0 mM, 3 mM, 6.25 mM, 12.5 mM, 25 mM and 50mM   -  Let the agar cool down before adding 3-AT, it is heat sensitiveMark and restreak 1-3 formed colonies from step 2 onto the interaction plates       suggestion: only use a small amount of cells for restreaking to reduce false positivesWrap parafilm around the platesIncubate plates at 30°C for 3-6 daysUse the lowest concentration of 3-AT that allows only small (   -  Possible concentrations of 3-AT: 0 mM, 3 mM, 6.25 mM, 12.5 mM, 25 mM and 50mM   -  Let the agar cool down before adding 3-AT, it is heat sensitiveMark and restreak 1-3 formed colonies from step 2 onto the interaction plates       suggestion: only use a small amount of cells for restreaking to reduce false positivesWrap parafilm around the platesIncubate plates at 30°C for 3-6 daysUse the lowest concentration of 3-AT that allows only small (       suggestion: only use a small amount of cells for restreaking to reduce false positivesWrap parafilm around the platesIncubate plates at 30°C for 3-6 daysUse the lowest concentration of 3-AT that allows only small (
?. [Growth assay]
Prepare plates: CSM -Leu -Trp (control) and CSM -Leu -Trp -His + 3-AT (interaction plate), use determined concentration of 3-ATRestreak 1 colony from step 2 onto the control plate and afterwards onto the interaction plate (reduces amount of cells and therefore false positives) Wrap parafilm around the platesIncubate plates at 30°C for 3-4 daysResult: Interaction of two proteins is positive if there is growth on both, the control and interaction plate, but without any growth of the controls on the interaction plate.
TITLE: HTBE ALI System Infection &amp; Sample Collections
AUTHORS: Briana L McGovern

[DESCRIPTION]
HTBE ALI System

[STEPS]
SECTION: Background on the HTBE ALI System
1. The air liquid interface (ALI) system provides a life-like experimental set up for human tracheal/bronchial epithelial cells. Cells are cultured (by Mattek) in a small circular insert. This insert is then gently placed, or optionally suspended, in a 6-well/12-well/24-well plate depending on the area of the insert.

The liquid interface:
Media in the wells of the plate is referred to as basal media. This basal media should be warmed before being plated. Basal media should be replaced every 2-3 days as the nutrients are depleted over time. 
Basal media is what will be manipulated in these HTBE ALI experiments. You can introduce drugs, antibodies, interleukins, etc into this basal media to treat the HTBE cells.

The air interface:
The actual area inside of the insert is known as the apical side, and is the air interface of the system. This apical surface generates mucus, much like the mucus of the airways. This mucus must be washed off of the surface every 2-3 days using TEER buffer or DPBS. You will also infect the cells using the TEER/DPBS. 
Apical washes will be collected and tittered at various time points of interest.

 

SECTION: Plating the inserts
2. Inserts will arrive embedded in an agar/gel to prevent movement during shipping. 
Once you receive the cells you should immediately move them to the incubator and prepare the plates that you wish to keep them in. 

The following is for a 12-well plate:
Warm the provided media and add 0.5-1ml to each well.
Disinfect a pair of tweezers using ethanol and gently lift an insert out of the gel/agar. Avoid having excess gel stuck to the insert. If gel is stuck on the insert gently remove it.
Move the insert to the well on the new plate*.
Repeat until all inserts are set in the new plates.
Incubate the plates overnight for acclimation.

*Mattek offers suspension set ups that float the insert in the well rather than have the insert placed in the well. We have not personally purchased with these pieces.
SECTION: Treatment via Basal Media
3. Using the provided media, set up your treatments.
I usually set up the treatments in 10ml/15ml tubes since you would want to make enough volume for triplicates.

 

ex) treating inserts on a 12-well plate with 3 concentrations (1:3 serial dilutions) of a drug
Conditions should be tested in triplicate at minimum and there should be both a DMSO (no treatment) control and a treatment control (for SARS-CoV-2 it would be Nirmatrelvir).

 

SECTION: Infection
4.
TITLE: Triaxial mechanical testing of dog colon samples
AUTHORS: Bhavesh Patel, Yanmin Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the step followed to conduct ex vivo triaxial mechanical testing (inflation, extension, torsion) of dog colon samples on our custom build testing platform. </div></div>

[STEPS]
?. [Collect tissue]
1. At termination of the animal, open the abdominal cavity to expose the colon. 2. Identify a ~20 cm colon segment and isolate it with sutures or clamps at both ends. 3. Take a picture with a scale (Pic.1).  4. Dissect the segment. 5. Gently remove luminal content by squeezing it out, and clean remaining content by flushing several times with saline until completely cleaned. 6. Position on a flat surface and take another picture with a scale (Pic.2). 7. Store the segment in Ca-free Kreb at 4°C until testing. 8. Conduct testing within 4 hours.
?. [Prepare triaxial machine for testing]
1. Fill tank and inflation liquid bottle with Ca-free Kreb.2. Open the 95% O2 – 5% CO2 line to aerate the Ca-free Kreb solution.3. Turn on the heater (setup for 37C) and the associate pump.4. Setup a pH-meter to monitor pH. Make sure pH = 7.4±0.1 before starting the test. Adjust pH if necessary.5. Calibrate camera using the known diameter of the tissue holder.6. Open main pressure valve. Open machine pressure valve to let Ca-free Kreb flow through and remove any air.
?. [Prepare tissue for testing]
1. Take tissue out of the storage solution and dissect connective tissues and major mesentery vessels.2. Mount the tissue on the triaxial machine and secure it with O-rings. 3. Open exhaust bolt on the tissue mount, then open the pressure valve and let solution flow through the tissue and out of the exhaust. 4. Close pressure valve and the exhaust bolt. 5. Once bath temperature is at 37C, start the test.
?. [Conduct testing]
1. Stretch the tissue back-end forth up to a 40% stretch five times while maintaining tissue pressure at 0.5 kPa and 0 twist.2. At multiples stretch steps where the tissue is not bulging, inflate-deflate the tissue back and forth four times between 0.5 and 2 kPa while maintaining the tissue at 0 twist.3. At multiples stretch steps where the tissue is not bulging and at multiple pressure steps between 0.5 and 2 kPa, twist the tissue back and forth four times between -40 and 40 degrees.
TITLE: Site directed mutagenesis by PCR
AUTHORS: Tobias von der Haar

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Design four primers in the following way: Primer 1 anneals upstream of the region to be mutatedPrimer 2 anneals downstream of the region to be mutated, in reverse direction, and has a 6 nt mismatch to the target at its 3'-end.Primer 3 anneals downstream of primer 2 (not overlapping with the primer 2 site).Primer M anneals of the site to be mutated, containing the mutation itself and 15 nt on either side of the mutation.If Primers 1, 2 and 3 are designed to anneal to vector sequences 5’ and 3’ of a multiple cloning site, they can be reused for any mutagenesis performed in this vector.
?. Perform two PCR reactions with your template DNA: the first with primers 1 and 2, the second with primers M and 3. You should use a proof-reading polymerase that does not append 'A' overhangs to the pCR product - if you use Taq polymerase, the nucleotide before the primer annealing site for primer M must be a T (this will allow base-peiring with the A residues appended to PCR products by Taq).Clean up the PCR products by resolbing them on an agarose gel, excising the bands, and using a gel extraction kit to retrieve the DNA.
?. Perform the second round PCR, using primers 1 and 3, and ~1 μl each of the two PCR producst from round 1 as template.In the initial rounds of the PCR, the two product will anneal and the product incorporating the mutation can be extended to full length - this contains annealing sites for both primers 1 and 3, and will be amplified in subsequent rounds. The other strand cannot be extended to full length due to the 3'-end mismatch.
?. Use the final PCR product for cloning  as for any other PCR product.
TITLE: Guion de entrevista en profundidad para profesorado sobre el impacto del método ECO en la docencia
AUTHORS: Juan Jesus  Torres-Gordillo, Nazaret  Tomé Miranda

[DESCRIPTION]
El propósito de esta entrevista es conocer el impacto de la implementación del método ECO en la docencia universitaria desde la perspectiva del profesorado. Los datos adquiridos serán analizados para ser utilizados en un informe final y posteriores publicaciones.
Se trata de una entrevista semiestructurada, que consta de 28 preguntas, divididas en cinco bloques: 6 preguntas sobre datos demográficos, 4 preguntas sobre sensaciones iniciales al implantar el método ECO por primera vez, 7 preguntas sobre conceptos, 7 preguntas en relación con los resultados obtenidos a través del método ECO y 4 preguntas finales sobre prospectiva. 

[STEPS]