eamag/protocols-with-steps huggingface dataset:
TITLE: GrownMD CBD Gummies (Pros and Cons) Is It Scam Or Trusted?
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[DESCRIPTION]
GrownMD CBD Gummies

[STEPS]
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How Does Grown MD CBD Gummies Work?

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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The aim of the current study was to investigate whether optimism and self-efficacy mediated the association between shyness and subjective well-being in a sample of Chinese working adults. Two hundred and eight participants completed the Revised Cheek and Buss Shyness Scale, Life Orientation Rest-Revised, Satisfaction with Life Scale, and Positive and Negative Affect Scale. Structural equation modeling results showed that optimism mediated the relationship between shyness and measures of subjective well-being (life satisfaction, positive and negative affect). Self-efficacy mediated the association between shyness and positive subjective well-being (life satisfaction and positive affect). These results suggest that optimism and self-efficacy play unique moderating roles in the relationship between shyness and subjective well-being. They also have important implications for the development of intervention programs aimed at promoting subjective well-being through enhancing self-efficacy and optimism.</p>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>this protocol describes the maintainance and treatment of RAW 264.7 macrophage cell lines with Mycobacterial PE/PPE proteins. It also describes estimation of secreted cytokines through ELISA. Estimation of level of IgG subclass from the serum is also described here.</p>

[STEPS]
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TITLE: SRB assay for measuring target cell killing
AUTHORS: Diana Rose E Ranoa

[DESCRIPTION]
The Sulforhodamine B colorimetric assay was used to measure effector CAR T cell killing of their target tumor cells. 

[STEPS]
1. Perform co-culture experiment of activated CAR T cells and target tumor cells at various tumor:effector ratio in a 96-well plate for 24 hours at 37'C, 5% CO2.
2. Remove cell culture supernatant 24 hours after co-culture assay.
3. Using a multichannel pipet, fix adherent tumor cells on the 96-well plate with 100uL cold 10% (w/v) tricholoroacetic acid (TCA) to each well, and incubate at 4'C for 1hr.
4. Wash four times by dipping entire plate in a basin with slow-running tap water. Tap plates on paper towels to remove excess water, and allow to dry at room temperature.
5. Add 100uL of 0.057% (w/v) Sulforhodamine B (SRB) solution to each well and incubate at room temperature for 30 minutes.
6. Immediately wash plates four times in a basin containing 1% (v/v) acetic acid to remove unbound dye.
7. After the plates have dried completely, add 200uL of 10mM Tris base solution (pH 10.5) to each well and incubate for 5 minutes with shaking.
8. Read plates in a microplate reader at A510nm.
9. Calculate the percentage of cell growth inhibition using the formula below:  
% cells killed = 100 - [mean OD(co-cultured sample)/mean OD(tumor cell only)]x100
TITLE: Electroporation of Agrobacterium
AUTHORS: Johannes Wolfram Debler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A quick guide on how to electroporate your plasmid of interest into electrocompetent </span><span style = "font-style:italic;">Agrobacterium tumefaciens </span><span>cells.</span></div></div>

[STEPS]
?. [Preparation of Electroporation mixture]
Get aliquotted electrocompetent Agrobacteria from -80°C freezer
{"blocks":[{"key":"6ojgv","text":"This protocol yields about 5 ml of electrocompetent Agrobacterium tumefaciens cells, aliquotted into 80 ul you get 50-60 tubes out of it.","type":"unstyled","depth":0,"inlineStyleRanges":[{"offset":52,"length":26,"style":"italic"}],"entityRanges":[],"data":{}},{"key":"11r7r","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":{}},{"key":"bpk1s","text":"Rifampicin is dark red and the colour will change over time, it does however NOT interfere with OD600 measurements, so you can use LB to blank. ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":{}},{"key":"8fgbc","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":{}}],"entityMap":{}}
?. [Preparation of Electroporation mixture]
add 0.1 - 0.5 µl plasmid to 80 µl Agro aliquot
?. [Preparation of Electroporation mixture]
mix gently and transfer into 1 mm electroporation cuvette
?. [Electroporation]
select Agrobacterium program on electroporator (check manufacturers handbook) and deliver pulse
?. [Regenerate electroporated cells]
flush cuvette with 500 µl SOC medium and transfer back to 1.5 ml tube
?. [Regenerate electroporated cells]
incubate at 28°C at 280 rpm for 3-4 hours
?. [Plating of electroporated cells]
transfer 20 µl into new 1.5 ml tube
?. [Plating of electroporated cells]
add 500 µl SOC
?. [Plating of electroporated cells]
plate 20 µl of SOC diluted Agro onto YM plates containing the correct antibiotics for your strain of Agrobacterium plus an antibiotic to select for your plasmid.
?. [Plating of electroporated cells]
incubate at 28°C for 2-3 days until colonies become visible
?. [Preparation]
Media:YM plates (For 1 litre: 10 g Mannitol, 0.4 g Yeast Extract, 0.5 g K2HPO4, 0.1 g NaCl, 0.2 g MgSO4 7H2O, 15 g Agar) --> autoclave and add appropriate antibiotics before making plates.SOB (For 1 litre: 0.5 g NaCl, 20 g Tryptone, 5 g Yeast Extract) --> autoclave2M Glucose solution (For 50 ml: 18 g Glucose) --> filter sterilizeSOC (For 100 ml: 99 ml SOB, 1 ml 2M glucose solution)Antibiotics:AGL1: rifampicin 100 ng/ulLBA 4404: rifampicin 100 ng/ul, streptomycin 100 ng/ul --> grows slow and clumpsGV3101::pMP90: rifampicin 100 ng/ul, gentamicin 25 ng/ul --> grows fastest of the threeAntibiotics:AGL1: rifampicin 100 ng/ulLBA 4404: rifampicin 100 ng/ul, streptomycin 100 ng/ul --> grows slow and clumpsGV3101::pMP90: rifampicin 100 ng/ul, gentamicin 25 ng/ul --> grows fastest of the three
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved <em>Drosophila melanogaster</em> transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.</p>

[STEPS]
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TITLE: CODEX Microtomy Tracking Sheet
AUTHORS: Leigh Propper, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Microtomy Tracking Sheet for CODEX Specimens</div><div class = "text-block">The process of sectioning CODEX specimens presents tediously at the tissue block trimming and microtomy stage.</div><div class = "text-block">As you begin to cut into a new or already used tissue block, you must keep track of how many microns of tissue are removed from the section during trimming, and also during microtomy.</div><div class = "text-block">Attached is a generic document that is used to keep record of what tissue block is being cut. All identification factors, trimming data, what tissue sections were used and their purpose, as well as storage information. </div><div class = "text-block">Formal logging of how far into the tissue block you have traveled as well as how many sections were used is an extremely important and necessary function in this process. </div></div>

[STEPS]
?.
TITLE: Brain Dissection of Post-natal Mice
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the procedure for removal and embedding of the post-natal mouse brain.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for making invertedClampFISH probes. </p>

[STEPS]
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TITLE: U Michigan - Cryoembedding
AUTHORS: Malcolm Low

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

[STEPS]
?. Procedure: Fixed Tissue 1.   Following fixation (immersion or perfusion), rinse tissue in phosphate buffer (PB, 0.1M, pH 7.3) containing 5, 10, and 20% sucrose for 24 hours per step or until the tissue "sinks” to the bottom of the container or tube. The final sucrose concentration in the tissue may vary from 5-30% depending on what conditions work best for the tissue in question. 2.   Cool 2 methyl-butane in a dry ice bath until dry ice no longer boils when added. 3.   Make embedding molds out of aluminum foil. Cut small squares, about 1” square or less. Use the bottom of a small glass vial or appropriately sized mold, form foil squares around bottom. 4.   While foil is still on the mold, write all information concerning the tissue going in the mold. 5.   Remove foil from mold, add OCT. 6.   Take tissue out of 20% sucrose and place in a small dish or weigh boat containing OCT to remove any excess sucrose. 7.   Place tissue in foil mold. Make sure all tissue is flat on the bottom of the mold. Cover tissue with OCT. 8.   Using forceps, hold mold in 2-methyl butane until OCT freezes. Try not to drop the mold in the 2-methyl butane, as the block will freeze too fast and crack. 9.   Keep in bucket on dry ice until all blocks are ready to be put in –20ºC or –80º C freezer.Fold excess foil over the top of the block to prevent freeze-drying. Procedure: Fresh Tissue Blocks of fresh tissue may be snap frozen at approximately -40º C and immediately sectioned on the cryostat or covered in OCT, snap frozen and stored at for later sectioning. The freezing temperature is critical, the slower the tissue freezes, the more ice crystals form. If the piece of tissue is very large there is a risk of it shattering if frozen too fast.
TITLE: Fixing Cell Pellets for Flow Cytometry
AUTHORS: Verity Goodwin, Emily Souster, Mathew Garnett, Fiona Behan, Charlotte Beaver, Rizwan Ansari, Adam Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed to outline the process of fixing cell pellets in 1.5ml tubes. It has been developed within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute.</div><div class = "text-block"><span style = "font-weight:bold;">Process diagram:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/11f7e279594fdb8947a627a79a011af77912ade8923993cae1dcb9e618960e5f/ccmmbdtbp.png" /></div></div>

[STEPS]
?. [Fixing]
In a fume hood, prepare a solution of 3.7% formaldehyde in PBS
Chemical safety: Formaldehyde 37% and 3.7% must be prepared and used only in the chemical fume hood, using chemical resistant gloves. Waste must be kept in the fume hood and disposed of appropriately.
?. [Fixing]
In the fume hood, tap the tube to loosen the pellet and resuspend each pellet in   of 3.7% formaldehyde. Mix well by pipetting up and down, ideally using a Rainin 1ml electronic pipette to obtain a single cell suspension and incubate at room temperate to fix for 10-20 minutes.
500 µl
?. [Fixing]
Centrifuge cells at  using a minifuge inside the fume hood.
Centrifuge: 300 34, 3 min
?. [Fixing]
Carefully remove the supernatent (dispose of waste formaldehyde appropriately) and resuspend the cell pellet in   PBS and transfer to a 5ml BD Falcon Round Bottom Polystyrene Tube by pipetting through its filtered cap.
500 µl
At this stage, the fixed cell suspension can either be stored at   overnight or analysed using flow cytometry immediately.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We aimed to compare the effectiveness of supraglottic airway devices as a conduit for unassisted tracheal intubation. We searched OVID-MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials, KoreaMed, and Google Scholar databases to identify all relevant randomized controlled trials (RCTs) of supraglottic airway devices as a conduit of tracheal intubation published until May 2017. The primary outcome was the overall success rate of intubation by the intention to treat (ITT) strategy. Secondary outcomes of the study were the overall success rate of tracheal intubation by the per protocol (PP) strategy, and the success rate of tracheal intubation at first attempt by ITT and PP. We conducted a network meta-analysis with a mixed-treatment comparison method to combine direct and indirect comparisons among supraglottic airway devices. Of 1396 identified references, 16 RCTs (2014 patients) evaluated unassisted intubation with supraglottic airway devices. Patients were allocated to LMA-CTrach, LMA-Fastrach, Air-Q, i-gel, CobraPLA, Aura-I or single-use LMA devices. Based on the surface under the cumulative ranking curve, the three best supraglottic airway devices for use as a conduit of unassisted tracheal intubation are LMA-CTrach(which includes video assisted tracheal tube guidance), single-use LMA-Fastrach, and LMA-Fastrach. LMA-Fastrach showed higher success rate of intubation compared with i-gel, CobraPLA, Air-Q and Ambu-Aura (RR 0.23, 95% CI 0.07 to 0.73; RR 0.18, 95% CI 0.048 to 0.67; RR 0.33, 95% CI 0.13 to 0.84; RR 0.075, 95% CI 0.011 to 0.50, respectively). However, the number of eligible RCTs was small which is a limitation of our study. Therefore, well-designed randomized trials performed in large patient populations are required in order to increase the confidence of the results</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the Immunostaining in fly NMJ boutons. It is from 'Flower Ca<sup>2+</sup> channel in CME and ADBE' of Yao CK et al.</p>
<p> </p>
<p>Please see the manuscript for the full method details.</p>

[GUIDELINES]
<p style="text-align: justify;">Primary antibody dilutions used: mouse anti-Dlg (mAb 4F3)[<a href="#_ENREF_57" target="_blank">57</a>], 1:100 (Hybridoma bank)[<a href="#_ENREF_58" target="_blank">58</a>]; mouse anti-Brp (nc82), 1:100 (Hybridoma bank)[<a href="#_ENREF_59" target="_blank">59</a>]; rabbit anti-GFP, 1:500 (Invitrogen); mouse monoclonal anti-HA, 1:200 (Sigma); rabbit anti-HA, 1:200 (Sigma); rabbit Cy3 conjugated anti-HRP, 1:500 (Jackson ImmunoResearch); guinea pig anti-Fwe-PB (GP100Y), 1:100. Secondary antibodies were diluted in 1:500 (Jackson ImmunoResearch and Invitrogen).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">To compare the staining intensity of boutons among different genotypes, larval fillets used in the same graph were stained in the same eppendorf. The images were captured using a Zeiss 780 confocal microscope, and the scan setup was fixed for the same experimental set. For data quantifications, single-plane confocal images were projected. The final staining intensity in boutons was calculated by subtracting background fluorescence intensity in the surrounding muscles from the staining intensity in boutons. The staining intensity of all type Ib boutons from the same muscles 6 and 7 in one image was averaged to obtain each data value. The dye unloading efficiency was calculated from F<sub>load</sub>-F<sub>unload</sub>)/F<sub>load</sub>. Images processing was achieved using LSM Zen and Image J.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p> </p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: UC Davis - Corticosterone RIA
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>The RIA (RadioImmuno Assay) is an assay method used for the quantification of various proteins. A standardized concentration of antibody specific to the analyte of interest is pipetted into test tubes.  A standardized concentration of the analyte of interest that is labeled with a radioisotope (usually</span><span style = "vertical-align:super;"> 125</span><span>I) is added to the tubes. Standards and samples are pipetted into the tubes and the tubes are incubated. During the incubation period the standardized concentration of labeled analyte and the unknown concentration of analyte in the samples will compete for binding sites on the antibody. After the incubation period a precipitating reagent is added to the tubes and the bound antibodies are precipitated out using the double antibody-polyethyleneglycol precipitation technique. The tubes are centrifuged and the supernatant is aspirated and the precipitate is counted (using a gamma counter if </span><span style = "vertical-align:super;">125</span><span>I is used). If there is a low concentration of the analyte of interest in the samples, the labeled analyte will have a higher probability for binding to the antibody and thus there will be a higher count in the precipitate. If there is a high concentration of the analyte of interest in the samples there will be a lower count of labeled analyte in the precipitate. The counts and known concentrations of the standards are used to generate a standard curve, and the counts of the samples are used to interpolate quantitative concentrations for the analyte of interest from the standard curve.</span></div></div>

[STEPS]
?. Dilute samples 1:200 with steroid diluent.
?. Add 0.3 ml diluent to NSB tubes and 0.1 ml to zero tubes.
?. Add 0.1 ml of calibrators and diluted samples.
?. Add 0.1 ml of tracer to all tubes.
?. Add 0.1 ml of antibody to all tubes except Total Count and NSB tubes.
?. Incubate at RT for 2 hrs.
?. Add precipitating reagent and centrifuge at 3000 rpm at 4°C for 30 min.
?. Aspirate supernatant without disturbing the pellet.
?. Count precipitate with gamma counter. Calculate standard curve and unknown values.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 1">
<p>Protocol for thymus dissociation (10-week old CD-1 female). </p>
</div>

[GUIDELINES]
<div title="Page 1">
<div>
<div>
<p><strong>Storage Conditions of Reagents </strong></p>
<p> </p>
<table>
<tbody>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p><strong>Reagent</strong></p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<p><strong>Storage Condition </strong></p>
</div>
</td>
</tr>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>DPBS (Thermofisher, 14190144)</p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<p>4°C</p>
</div>
</td>
</tr>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>0.5 M EDTA (Ambion, AM9260G)</p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<div title="Page 1">
<div>
<div>
<div>
<p>room temp.</p>
</div>
</div>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>BSA (Sigma, A8806)</td>
<td>4°C</td>
</tr>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>Protease from <em>Bacillus Licheniformis </em>(Sigma, P5380)</p>
</div>
</div>
</div>
</div>
</td>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>Store 100 μL aliquots (100 mg/mL) in DPBS at -80°C</p>
</div>
</div>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div title="Page 1">
<div>
<div>
<div>
<p>DNAse 1 (Applichem, A3778)</p>
</div>
</div>
</div>
</div>
</td>
<td> Store 10 μL aliquots (250 U/10 μL) in DPBS at -80°C</td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong><em>Bacillus Licheniformis</em> enzyme mix (1 mg/mL enzyme): </strong></p>
<p>492 µL DPBS (No added Ca, Mg)<br /> 0.5 mM EDTA (0.5 μL of 0.5 M EDTA/mL)<br /> 125 U DNAse 1 / mL (2.5 µL)</p>
<p>5 µL of 100 mg/mL enzyme (final conc. 1 mg/mL)</p>
<p>--------------------------------------------<br /> <strong><em>Bacillus Licheniformis</em> enzyme mix (2 mg/mL enzyme): </strong></p>
<p>487 µL DPBS (No added Ca, Mg)</p>
<p>0.5 mM EDTA (0.5 μL of 0.5 M EDTA/mL)<br /> 125 U DNAse 1 / mL (2.5 µL)<br /> 10 µL of 100 mg/mL enzyme (final conc. 2 mg/mL)</p>
<p>--------------------------------------------<br /> +12.5 mg of tissue</p>
</div>
</div>
</div>

[STEPS]
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