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

[GUIDELINES]
<p><strong>Application Notes </strong></p>
<p> </p>
<p>1. Activated cell populations can be prepared from in vivo-stimulated tissues or from in vitro-stimulated cultures (e.g., antigen-specific activation or mitogen-induced). For cytokine and chemokine detection, it is critical to include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No. 420601) or monensin (BioLegend Cat. No. 420701) in the last 4-6 hours of cell culture activation. The cells can be suspended and distributed to 12 x 75 mm plastic tubes or microwell plates for immunofluorescent staining.</p>
<p> </p>
<p>2. Different cytokines/chemokines have different production peaks. In order to obtain optimal staining signals, the stimulation conditions for each stimulant need to be optimized.</p>
<p> </p>
<p>3. Some antibodies recognizing native cell surface markers may not bind to fixed/denatured antigens. For this reason, it is recommended that staining of cell surface antigens be done with live, unfixed cells PRIOR to fixation/permeabilization and staining of intracellular targets. Altering the procedure such that cells are fixed prior to staining of cell surface antigens requires that paraformaldehyde-denatured antigen reactive antibody clones be empirically identified.</p>
<p> </p>
<p><strong>Note</strong></p>
<p> </p>
<p>To confirm specific anti-cytokine staining, a blocking experiment is recommended in which cells are fixed/permeabilized then preincubated with an excess amount of unlabeled anti-cytokine antibody and/or the recombinant cytokine of interest is preincubated with fluorophore-conjugated anti-cytokine antibody before its addition to the cells.</p>
<p> </p>
<p><strong>Related Information</strong></p>
<p><br />1. Assenmacher, M., et al. 1994. Eur. J. Immunol. 24:1097.<br />2. Elson, L.H., et al. 1995. J. Immunol. 1995. 154:4294.<br />3. Jung T, et al. 1993. J. Immunol. Methods 159:197.<br />4. Prussin C., et al. 1995. J. Immunol. Methods 188:117.<br />5. Vikingsson A., et al. 1994. J. Immunol. Methods 173:219.</p>
<p> </p>
<p><strong>Reagent List</strong><br />1. Cell Staining Buffer (BioLegend Cat. No. 420201)<br />2. Monensin (BioLegend Cat. No. 420701)<br />3. RBC Lysis Buffer (BioLegend Cat. No. 420301)<br />4. Brefeldin A (BioLegend Cat. No. 420601)<br />5. Fixation Buffer (BioLegend Cat. No. 420801)<br />6. Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002)<br />7. Cyto-Last™ Buffer (BioLegend Cat. No. 422501)</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Preparation  of yeast-malt-sucrose-agar, that is used as solid media for growing <em>Zt.</em> This is called 'YMA' in Bruce McDonald lab, althoug the same acronym is used also for different media.<br /><br />Have a look for example: Zain, M. E., et al. 'Influence of growth medium on diagnostic characters of Aspergillus and Penicillium species.' <em>African Journal of Microbiology Research</em> 3.5 (2009): 280-286.</p>

[STEPS]
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TITLE: Live-cell imaging: Mitochondria membrane potential
AUTHORS: gurvir.virdi, mineechoi

[DESCRIPTION]
Mitochondrial membrane potential is the electrogenic potential between the inner membrane and matrix of mitochondria which, in combination with the mitochondrial pH gradient, provides the force to drive protons into mitochondria to generate ATP. 
This protocol presents instructions on how to measure mitochondrial membrane potential in live-cell imaging.   

[STEPS]
1. Cells were washed 2x with HBSS (Invitrogen)
2. They are then incubated w 25 nM tetramethylrhodamine methyl ester (TMRM, Thermo Fisher Scientific) in HBSS for 40 min at Room temperature.
 


3. After 40 minutes, confocal microscope imaging is performed in the presence of TMRM (in HBSS); the 560 nm laser was used to excite, and fluorescence was measured above 580 nm.
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 0.1-0.2% of laser output to prevent phototoxicity, and the pinhole is set to allow optical slice at approximately 1-2 μm. Z-stack images are collected. 
4. For dynamic TMRM a time-series with 5 second intervals was performed on the same confocal microscopy set up.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Diet-induced obesity by administration of a western-type diet high in fat and sugar to mice may result in an elevation of plasma total and LDL-cholesterol levels, triglycerides and the levels of the liver enzymes alkaline phosphatases and aspartate (AST) and alanine aminotransferases (ALT).</p>

[GUIDELINES]
<p>-The acid citrate solution is best kept at +4°C and made fresh every month.</p>
<p>-Concentrations of plasma metabolic parameters need to be multiplied by the dilution factor 3.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how the parasitic flatworms (S. solidus) were cultured in the lab for:<br /><br /><em><span style="color: #222222; font-family: arial, sans-serif; font-size: 12.8px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 1; word-spacing: 0px; -webkit-text-stroke-width: 0px; display: inline !important; float: none; background-color: #ffffff;">Hebert, F, O; Grambauer, S; Barber, I; Landry, C, R; Aubin-Horth, N (2016): Reference transcriptome sequence resource for the study of the Cestode Schistocephalus solidus, a threespine stickleback parasite. GigaScience Database. </span><a href="http://dx.doi.org/10.5524/100197" target="_blank">http://dx.doi.org/10.5524/<wbr />100197</a></em>

[BEFORE_START]
Before culturing worms, culture tubes need to be set up with membrane and autoclaved and RPMI media made up and autoclaved. <br />• Cut a piece of dialysis membrane twice the length of the tube. Make into a U shape to fit inside the tube (do not fold the membrane as the crease can affect the set up) leaving 1-2 cm folded over the top on each side and screw the lid on. Wrap in foil and autoclave.<br />• Fresh RPMI media should be prepared each time you culture worms. Weigh out the appropriate quantity of powder (10.4g/L) and make up to the required volume in a 400ml Duran bottle with ddH20 and autoclave

[GUIDELINES]
<strong>Equipment required:</strong><br />
<ul>
<li>Culture Tube - Tube culture screw cap borosilicate glass phenolic cap with PTFE liner use up to 121°C 26mm x 200mm Pyrex from Fisher product code 12349339</li>
<li>Dialysis Membrane - 30m SIZE 1 DIALYSIS TUBING 12-14000 DALT from Medicell product code DVT.12000.01</li>
<li>RPMI Media – RPMI-1640 Medium , With L-glutamine, without sodium bicarbonate, powder from Sigma product code R6504-10X1L</li>
<li>Horse Serum -Horse Serum Heat inactivated sterile-filtered from Sigma product code H1138-6X500ML</li>
<li>Penicillium - HyClone; Pen/Strep/Glutamine from Fisher product code 12340243</li>
</ul>
<strong>Before culturing worms:<br /></strong>
<ul>
<li>Culture tubes need to be set up with membrane and autoclaved and RPMI media made up and autoclaved.</li>
</ul>
<ul>
<li>Cut a piece of dialysis membrane twice the length of the tube. Make into a U shape to fit inside the tube (do not fold the membrane as the crease can affect the set up) leaving 1-2 cm folded over the top on each side and screw the lid on. Wrap in foil and autoclave.</li>
</ul>
<ul>
<li>Fresh RPMI media should be prepared each time you culture worms. Weigh out the appropriate quantity of powder (10.4g/L) and make up to the required volume in a 400ml Duran bottle with ddH20 and autoclave</li>
</ul>

[STEPS]
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TITLE: FUNDIS DNA Extraction, PCR, Oxford Nanopore Technologies MinION Sequencing and data analysis. 
AUTHORS: Harte Singer, Stephen Douglas Russell

[DESCRIPTION]
Generating DNA barcode sequences from fungal samples using the Oxford Nanopore Technologies MinION platform. Some methods adapted from Stephen D. Russell's protocols dx.doi.org/10.17504/protocols.io.36wgq7qykvk5/v3

[STEPS]
TITLE: Sterilizing and Preparing Seeds for Laboratory Use
AUTHORS: Gabriela Paredes

[DESCRIPTION]
Sterilizing seeds is a critical step in many plant biology and microbiology experiments to ensure that the seeds are free from contaminants such as bacteria, fungi, and other microorganisms. This protocol outlines the steps for effectively sterilizing seeds using a 25% commercial bleach solution, followed by thorough rinsing with sterile distilled water. This process ensures that the seeds are clean and ready for subsequent experimental treatments.

[GUIDELINES]
Ensure Thorough Mixing: Proper agitation during the sterilization and rinsing steps is crucial to ensure even contact of the solution with all seeds.
Sterile Handling: After the sterilization process, handle the seeds only with sterile equipment and in a sterile environment to maintain their sterility.
Check for Residual Moisture: Make sure the seeds are thoroughly dried before storage or use to prevent fungal growth or other contamination.

[STEPS]
SECTION: Prepare the Bleach Solution:
1. Prepare a 25% bleach solution by mixing 250 ml of commercial bleach with 750 ml of sterile distilled water to obtain a total of 1000 ml of the solution.
Ensure that the solution is prepared in a sterile container.
SECTION: Sterilize the Seeds:
2. Place the counted seeds into an autoclaved Erlenmeyer flask. Ensure that the seeds are evenly distributed at the bottom of the flask.
SECTION: Sterilize the Seeds:
3. Add 150 ml of the prepared 25% bleach solution to the flask, ensuring that the solution fully covers the seeds.
SECTION: Sterilize the Seeds:
4. Place the flask on a magnetic stirrer or shaking platform and agitate the solution for 10 minutes to ensure thorough contact between the bleach solution and the seeds.
SECTION: Rinse the Seeds:
5. After 10 minutes, carefully decant the bleach solution into a waste container suitable for bleach disposal.
Add twice the volume of sterile distilled water to the flask containing the seeds and agitate gently for 3 minutes to rinse off the bleach.
Decant the rinse water and repeat this rinsing process three times to ensure all bleach residues are removed.
SECTION: Remove Seed Coats (Optional):
6. If the experiment requires seeds without seed coats, this can be done after the rinsing process.
Wearing sterile gloves, gently rub the seeds between your fingers or use autoclaved forceps to remove the seed coats.
SECTION: Dry the Seeds:
7. Transfer the sterilized seeds to a sterile Petri dish labeled appropriately.
Place the Petri dish in a laminar flow hood or other sterile environment and allow the seeds to dry for at least 30 minutes. This step helps reduce moisture on the seed surface, minimizing the risk of contamination during subsequent handling.
SECTION: Storage or Further Use:
8. If the seeds are not to be used immediately, store them in a dry, sterile, airtight container. Keep the container in a sterile environment to prevent contamination until the seeds are needed.
TITLE: Immunocytochemistry Staining Protocol
AUTHORS: Sam Li

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

[STEPS]
?. [Sterilization (for 12-well plates with coverslips):]
Transfer a single cover slip into a 12-well plate. Then add 1 mL of 70% Ethanol into a well for 20 minutes at room temperature.
?. [Sterilization (for 12-well plates with coverslips):]
Wash quickly three times with PBS.
?. [Poly-Lysine Coating for 12-Well Plates (optional; for loosely attached cells):]
Add 1 mL of 0.1 mg/mL Poly-D-lysine solution into a well for 15 minutes at room temperature.
?. [Poly-Lysine Coating for 12-Well Plates (optional; for loosely attached cells):]
Wash quickly three times with PBS and let dry before plating cells.
?. [Sample Preparation:]
Grow cultured cells on cover slips or in wells overnight at 37°C. At the time of fixation, cells should be ~70-80% confluent in single layer.
?. [Sample Preparation:]
Rinse cells briefly in PBS.
?. [Sample Preparation:]
Fix cells by incubation with freshly made 1% Paraformaldehyde in PBS for 10 minutes at room temperature.
?. [Sample Preparation:]
Rinse three times quickly in PBS.
?. [Sample Preparation:]
For intracellular staining, add permeabilization solution and incubate at room temperature for 10 minutes. Then wash quickly three times in PBS.
?. [Sample Blocking:]
Block samples in 1 mL of blocking buffer at room temperature for 30 minutes.
?. [Sample Staining:]
Dilute the primary antibody to the recommended concentration/dilution in blocking buffer.
?. [Sample Staining:]
For 8-well chamber slides, add 200 µL per well. For 12-well plates, add 500 µL per well. Incubate two to three hours at room temperature or overnight at 4°C. If using conjugated antibodies, perform this step in the dark.
?. [Sample Staining:]
For surface staining, rinse 3 times quickly in PBS. For intracellular staining, quickly wash once followed by incubation with wash buffer for 5-10 minutes. Then quickly wash an additional two times.Note: If using primary antibodies directly conjugated to fluorochromes, then skip to step 13.
?. [Sample Staining:]
Prepare fluorochrome-conjugated secondary antibody in blocking buffer according to the manufacturer’s specification data sheet, and add 200 µl per well to the 8-well chamber slides. For 12-well plates, add 500 µL per well.
?. [Sample Staining:]
Incubate the samples for one hour, at room temperature, in the dark.
?. [Sample Staining:]
For surface staining, rinse three times quickly in PBS. For intracellular staining, quickly wash once followed by incubation with wash buffer for 5-10 minutes, then quickly wash an additional two times.
?. [Sample Staining:]
Optional: To stain F-actin, prepare a working solution of Flash Phalloidin™ by diluting it 1:20-1:100 in PBS. Add 200 µL per well for an 8-well plate or 500 µL per well for a 12-well plate. Stain for 20 minutes at room temperature in the dark.
?. [Sample Staining:]
Apply anti-fade mounting medium to the cover slip.
?. [Sample Staining:]
Seal slides with nail polish.
TITLE: Scintillation Count of radiolabeled whole cell
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scintillation Count of radiolabeled whole cell</div></div>

[STEPS]
?. [Steps]
Grow OG1RF to mid log phase in BHI.
?. [Steps]
Harvest 5mL of cells.
?. [Steps]
Wash twice in 1X davis salts without glucose.
?. [Steps]
Re-suspend in 5mL 1X davis salt without glucose with 10mg/mL BSA.
?. [Steps]
Incubate at 37oC for 30 minutes as starvation treatment.
?. [Steps]
Move to hot room.
?. [Steps]
Place cells in 37oC heat block for assay  or in 95oC heat block for certain death.
?. [Steps]
Add the hot/cold mix to 5mL of cells in the davis/BSA buffer and start the timer.
?. [Steps]
Immediately take the 200ul of cells out for T0 - there are placed on the filter paper which is connected to a vacuum - these are washed with 2 mL of wash buffer (1X davis salts with 10mg/ml BSA).
?. [Steps]
Stager the time points every 5 minutes for about 20-25 minutes. Then , repeat this for the heat killed cells.  Note that there isn't any difference between the heat killed cells and the 37oC cells. We are still working on control for this part of the assay.
?. [Steps]
At completion of the assay filters are added to vials with scintillation fluid and counted by the machine.
TITLE: Recombineering
AUTHORS: Elizabeth Fozo

[STEPS]
?. [Template]
Prepare a PCR product. Typically, 200-600 ng/transformation. See note below. If using a plasmid template, may have to digest using DpnI or another enzyme to remove the additional plasmid
?. [Template]
Following purification, combine PCR products and ethanol precipitate. Resuspend products in water
I usually combine 2-4 PCR products into 5 ul and use 2.5 ul for a first recombineering attempt.
?. [Strains]
Grow up 25 ml of a strain of choice at 30 or 32 degrees (do NOT use DY330. Use NM400, NM1100, or some such derivative).
?. [Strains]
When OD600 approximately 0.4, transfer to 42 degrees shaking water bath for 15 – 20 minutes.
?. [Strains]
After 15-20 minutes, place the flask in an icy slurry and swirl. Keep in icy slurry for about 2 minutes.
?. [Strains]
Spin at 4 degrees, 10 minutes, 4150 rpm.
?. [Strains]
Wash cells with 1 ml cold 10% glycerol at 4 degrees, three times
?. [Strains]
Resuspend cells in   cold 10% glycerol.
100 µl
?. [Strains]
Electroporate: use   of cells + DNA. Immediately after electroporation, add 950 ul of SOC. Shake at 30 degrees for 1 hour. Plate 1/10 onto one plate, and concentrate the remaining 900 ul and plate to a second plate.
50 µl
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>These were developed to measure potential microbial activity for uptake of acetate and bicarbonate by microbes in venting hydrothermal fluids. The protocol was designed for Axial Seamount diffusely venting fluids, but can be applied to other fluid systems (e.g. cold, oxic fluids, seawater, etc). Concentrations of label will depend on environment of interest.</p>

[BEFORE_START]
<p>This protocol assumes you have already collected the sample (seawater, vent fluid, etc) of interest.</p>

[STEPS]
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TITLE: Electrotransformation of Clostridium species
AUTHORS: Chin Yee Tan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A brief protocol for electrotransformation of Clostridium species</div></div>

[STEPS]
?. Inoculate   with  overnight.
[BHI-supplemented broth]
[stock culture]
?. Use the overnight culture to inoculate  to a starting density of OD 0.02.
[BHI-supplemented broth]
?. Harvest early-exponential phase culture (OD 0.2 to 0.25) by centrifugation by 12,000g x  at.
?. Wash once in .
[SML electroporation buffer]
?. Resuspend in  .
[SMP electroporation buffer]
?. Take  of aliquots and and mix with  of DNA, transfer to prechilled cuvettes with 0.2cm gap.
400 µl
500 ng
?. Incubate on ice for .
?. Electroporate at the following parameters: 25μF, resistance 200Ω, voltage 1.8kV.
?. Immediately transfer the cells into  of BHI broth and incubate for .
10 ml
?. Plate cells in dilutions on solid selective and non-selective BHI agar.
TITLE: T4 Ligation
AUTHORS: Carolina Lopez

[DESCRIPTION]
Protocol for DNA ligation using T4 ligase

[STEPS]
SECTION: T4 Ligation Protocol (Thermofisher, 15224041):
2. 1. For initial reaction, Mix: 
 
X and Y should be calculated for 3 Insert:1 vector molar ratio

Example of Molar Ratio calculation for 3000bp vector with 500bp PCR product insert:

 


2. Incubate at room temperature for 15 min.
3. Transform Product into E. coli
        a) Add 2 µL of product to 50 µL of TOP10 cells and pipette up and down slowly to mix.
        b) Incubate for 20 min on ice.
        c) Heat shock bacteria in 42 °C waterbath for 1 min.
        d) Incubate on Ice for 3 min.
        e) Add 100 µL of SOC media and shake in warm room for 60 min.
        f) Plate bacteria onto LB-Antibiotic Plate and spread cells with plate spreader to get individual colonies.
        g) Incubate overnight in 37 °C warm room.
        h) Pick colonies for miniprep growth and sequencing.
SECTION: T4 Ligation Protocol (Thermofisher, 15224041):
1. T4 DNA Ligase catalyzes the joining of two cohesive- or blunt-ended strands of DNA between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides.
TITLE: Expressão de scPPx 1 
AUTHORS: Mariana Jacques

[STEPS]
?. [:]
Tampão C -60 mM Tris HCl pH 7,5, 6 mM MgCl2, Glicerol 30%.Tampão B- 60 mM Tris HCl pH 7,5, 6 mM MgCl2, 250 mM ImidazolTampão de Lise - ? mM Tris HCl pH 7,5,  200 mM NaCl, 5% GlicerolTampão A- 60 mM Tris HCl pH 7,5, 6 mM MgCl2, 10 mM Imidazol.
?. [:]
Cultura em 5mLCultura em 1 L
?. [:]
Crescer até 0,6 - 0,8 DO 600 ( Guardar 20 μL)
?. [:]
Induzir com 500 μM de IPTG por 3 h/ 37 °C
?. [:]
Centrifugar (____ min/_____ g) e ressuspender em 30 mL
?. [:]
Lisar ( Potência 30%/  10 pulso de 10 seg a cada 1 min)
?. [:]
Spin 5000 g/ 10 minPassar em filtro de 0.8 μm/  0,4 μmCentrifugar para baixar o debris celular, passar em filtro de 0,22 μm
?. [Colunas ]
Coluna de Níquel 20 ml Tampão A5 mL Tampão A8,5 ml Lisado5 mL Tampão B
?. [Colunas ]
Coluna de Desalting 25 ml Tampão C1,5 ml Amostra  2 ml Tampão C ( Recolher)(Repetir esse processo 3 vezes)25 ml Tampão C1,5 ml Amostra
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is suitable for rapidly generating extracts of fungal cells for gel electrophoresis or western blots. It was optimised for complete extraction of difficult-to-solubilise proteins. This protocol was originally described in <a href="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001078" target="_blank">von der Haar 2007</a>.</p>

[STEPS]
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TITLE: Denaturation analysis of α-synuclein fibrils
AUTHORS: Arpine Sokratian

[DESCRIPTION]
This protocol details the specific parameters used to accurately measure the denaturation sensitivity of α-synuclein fibrils. Essential parts of this protocol describe the evaluation of sonicated fibrils to ensure the reproducibility of the results. Visualization of α-synuclein is recommended to be conducted using slot-blot analysis with aggregate-specific α-synuclein antibodies.

[STEPS]
SECTION: Preparation of α-synuclein sonicated fibrils
1. Follow the protocol to prepare the batch of sonicated fibrils. If the samples have been prepared earlier and kept at -80, thaw down in a water bath. Then, perform a quality control check to ensure the size distribution and concentration are corresponding to values outlined in the protocol attached.
 


SECTION: Preparation of α-synuclein sonicated fibrils
2. Evaluated sonicated fibrils should be diluted in PBS to concentration of 5 mg/mL. 
SECTION: Preparation of denaturating agents
3. Prepare stock solutions of 
6M Guanidine HCL   
10% SDS -    
10% Sarkosyl -   

SECTION: Preparation of denaturating agents
4. Dilute the chemical solutions: 
6M, 2M, 1M, 0.5M, 0.25M, 0.125M for GuHCL
2%, 0.2%, 0.02%, 0.002%, 0.0002%, 0.00002%
8%, 4%, 2%, 1%, 0.5%, 0.25%

SECTION: Reaction mix preparation
5.  
Calculate the reaction mix: 2 mg/mL concentration of α-synuclein in 50 µL in reaction mix
Use protein low-binding tubes: 

Reaction mix: 25 uL of chemical solution + 5 uL of PBS + 20 uL of 5 mg/mL sonicated fibrils
Once added, mix up very well 
Prepare reaction mixes for each concentration of chemical solution in triplicates
SECTION: Reaction mix preparation
6. Incubate at room temperature for 30 min
SECTION: Reaction mix preparation
7. Evaluate the denaturation process using DLS analysis


 

SECTION: Reaction mix preparation
8. Dilute samples up to 200 ng/mL (10.000x) to reach 1 mL of the volume, vortex before each dilution cycle
SECTION: Reaction mix preparation
9. Follow instruction for the slot-blot analysis, 
use    at concentration 1:5000 to visualize aggregated forms of a-synuclein species after treatment with chemical compounds
TITLE: Prepare sodium azide
AUTHORS: Andreas Sagen

[DESCRIPTION]
Prepare sodium azide solution

[STEPS]
SECTION: 50 mL 10% sodium azide
1. In a 50 mL tube, fill with 40 mL endotoxin-free water
SECTION: 50 mL 10% sodium azide
2. Empty a full bottle of 5 g Sodium azide powder in the tube

Materials:
 

 

SECTION: 50 mL 10% sodium azide
3. Mix powder completely, then top-up to 50 mL endotoxin-free water
SECTION: 50 mL 10% sodium azide
4. Filter solution with a 0.22 µm pore-size filter