eamag/protocols-with-steps huggingface dataset:
TITLE: Immunocytochemistry for the characterization of hiPSC to Motor Neuron differentiation
AUTHORS: Mallory Wright, ckremitz, William J Buchser

[DESCRIPTION]
This immunocytochemistry protocol is used for the characterization of IPSC differentiation into motor neurons using several biomarkers: neuroepithelial cells (SOX1), motor neuron progenitors (OLIG2 and NKX2.2), motor neurons (MNX1), and the mature motor neurons (ISL, ChAT, MAP2).

*Primary and secondary antibody information located in materials section
 





[BEFORE_START]
To ensure that the observed results are not just random events, use controls, such as undifferentiated IPSCs when doing immunocytochemistry. 

Here is an example of how we seed cells onto a 96-well plate. The plate has an equal amount of wells of differentiated (blue) and undifferentiated cells (yellow) for analysis, and we always include a moat (evaporation barrier). The cells are also counted and seeded at equal densities.

 


[STEPS]
1. Remove the medium from your cells 
 





2. FIX - Dilute 32% Paraformaldehyde solution to 4% PFA in 1X phosphate-buffered saline (PBS) 
3. Add 100uL of 4% PFA to each well in the 96-well plate. (1ml if using a 6-well plate). Incubate for 15 min at room temperature.

4. Remove the fixative solution and wash with 1XPBS at 100ul per well using a multichannel pipette. Repeat 3 times.
 

5. PERMEABILIZE - Add 100uL of 0.5% Triton X-100 to each well of a 96-well (1ml if using 6-well plate)
6. Incubate for 15 min at room temperature.

7. Remove the permeabilization solution and wash 3 times with 1XPBS 
8. BLOCK - Add 100ul of 3% BSA (bovine serum albumin) or blockAid-blocking solution to each well of a 96-well plate slowly to Block. (1ml if using 6-well plate)
9. Incubate for at least60 min (up to overnight) at room temperature.

10. Calculate the amount of primary antibody needed (located in materials section) and dilute in 3% BSA + 0.3% Triton X-100 solution 
11. PRIMARY ANTIBODIES - Remove 3% BSA from wells and add 100uL of primary antibody per well

 

12. Incubate for 60 min at room temperature in a dark place or overnight at 4°C.    
(Different primary antibody stains may take longer to stain and could require optimization).
13. Remove primary antibody and wash three times slowly with 1xPBS 
 

14. SECONDARY ANTIBODIES - Calculate amount of secondary antibody needed (located in materials section) and dilute in 3% BSA+ .3% TritonX-100 solution and 1:4000 Hoechst. 
15. Add 100uL per well in 96-well and incubate for at least 60 min at room temperature in a dark place.  

16. Remove secondary antibody and wash three times with 1xPBS 
17. Scan on the confocal microscope
TITLE: ssDNA Extraktion
AUTHORS: Franziska Patzold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ssDNA Extraction Protocol</div><div class = "text-block"><span>This protocol is based on the Oligonucleotide extraction protocol of the NEB PCR & Clean-up Kit and the Qiagen DNeasy Protocol with a few modifications to maximize DNA output. First results of this protocol will be published soon, including first sequencing results of the so extracted DNA. Please understand that this protocol is so far only tested for tissue (legs and abdomen) of invertebrates (Lepidoptera, genus </span><span style = "font-style:italic;">Hyles</span><span>). I recommend testing this protocol with tissue of non valuable specimens first. </span></div></div>

[STEPS]
?. [Elution]
Place dry tissue in 1.5 ml microcentrifuge tube and grind it, using an oscillating mill (MM 400, Retsch) for 1 min at a frequency of 25 Hz, using two sterile stainless steel balls with a diameter of 3 mm
?. [Lysis]
Insert to the ground tissue mix well by handshaking, don´t vortex! and gently spin down the solution.
[Lysis buffer]
[Proteinase K]
[total volume]
?. [Lysis]
Put the tube in an Mixing Block (MB-102, Thermocell)Mix gently, and spin again.
56 °C
[ at least, better overnight]
?. [Extraction]
Addto themix by pipetting up and down.
[DNA Cleanup Binding Buffer]
[sample]
?. [Extraction]
AddMix well by pipetting up and down or flicking the tube. Do not vortex.
[ethanol (≥ 95%)]
?. [Extraction]
Insert column into collection tube,load sample onto column and close the cap.then discard flow-through
[Centrifuge]
?. [Extraction]
Re-insert column into collection tube.AddandDiscard flow-through.
[DNA Wash Buffer]
[Centrifuge]
?. [Extraction]
Repeat Step 7.This step is recommended for removal of  enzymes that may interfere with downstream applications(e.g.,  Proteinase K).
?. [Extraction]
Transfer column to a clean 1.5 ml microfuge tube.Use care to ensure  that the tip of the column does not come into contact with the  flow-through.If in doubt, re-spin for 1 minute to ensure traces of salt  and ethanol are not carried over to the next step.
?. [Two-Step-Elution]
Addto the center of the matrixWait forthen spin forto elute the DNA.
[Elution Buffer]
[Incubation]
[Centrifuge]
?. [Two-Step-Elution]
Repeat Step 10 to extract all DNA
TITLE: Ethical Considerations and Informed Consent (Part 11 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Part 11 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: (Mick&#39;s) Trial K+ minimal Media Recipe, 1_16_2019
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Trial K</span><span style = "vertical-align:super;">+ </span><span>minimal Media Recipes</span></div></div>

[STEPS]
?. [5X M9 Media without K]
33.9g/L Na2HPO45g/L NH4Cl2.5g/L NaCl15g NaH2PO4 (was KH2PO4)
?. [1L of Trial K+ Minimal Media]
1. Add 200mL 5X M9 to 500mL MiliQ water.1mL of 1M MgSO4   -   1mM final0.1mL of 10mg/mL Thiamine   -   1ug/mL5mL of 80% glycerol    -    0.4% final5mL of 10mg/mL AA mix 1 - 50ug/mL final5mL of 10mg/mL AA mix 2 - 50ug/mL final0.1mL of 1MCaCl     -    0.1mM final500mg AA mix 3Check pH2. Fill with MiliQ water to 1L3. Filter with 0.2-micron filter
?. [250mL of Trial K+ Minimal Media]
1. Add 50mL 5X M9 to 150mL MiliQ water0.25mL of 1M MgSO4   -   1mM final0.025mL of 10mg/mL Thiamine-1ug/mL final1.25mL of 80% glycerol    -    0.4% final1.25mL of 10mg/mL AA mix 1-50ug/mL final1.25mL of 10mg/mL AA mix 2-50ug/mL final0.025mL of 1MCaCl     -    0.1mM final125mg AA mix 3Check pH2. Fill with MiliQ water to 250mL3. Filter with 0.2-micron filter
TITLE: RAD-sequencing
AUTHORS: Tomasz Suchan

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

[STEPS]
?. [Preparation]
Prepare reagents
?. [Restriction reaction]
Prepare the restriction reaction mix for all the samples using the amounts of reagents below:
[nuclease-free water]
[CutSmart buffer (10x)]
[MseI (10,000 U/ml)]
[SbfI (HF) (20,000 U/ml)]
?. [Ligation reaction]
Combine 9 uL of the restriction reaction product with 1 uL of the P1 adapter.
Remember to use a unique P1 adapter for each sample!
?. [Purification]
Purify the reaction using standard AMPure XP protocol using 0.8x ratio of AMPure and elute in 20 uL of nuclease-free water or 10mM Tris
?. [Library amplification]
Prepare the PCR reaction mix for all the samples using the amounts of reagents below:
[nuclease-free water]
[Q5 buffer (5x)]
[dNTPs (25 mM)]
[Forward primer (5 uM)]
[Reverse primer (5 uM)]
[Q5 Hot Start High-Fidelity polymerase]
?. [Restriction reaction]
Incubate 3 h at 37°C, heat-kill enzymes for 20 min at 65°C
[restriction reaction]
[restriction reaction]
[enzyme inactivation]
[enzyme inactivation]
?. [Restriction reaction]
Combine 6 uL of DNA with 3 uL of the restriction mix.
?. [Ligation reaction]
Prepare the ligation reaction mix for all the samples using the amounts of reagents below:
[CutSmart buffer (10x)]
[ATP (100 mM)]
[P2 adapter]
[T4 DNA ligase (400,000 U/ml)]
[nuclease-free water]
?. [Ligation reaction]
Add 1.6 uL of the ligation mix to each sample.
?. [Ligation reaction]
Incubate 3 h at 16 °C
[ligation reaction]
?. [Library amplification]
Combine 6 uL of purified ligation product with 14 uL of the PCR mix.
?. [Library amplification]
PCR program:30 s at 98°C20cycles: 20s at 98°C, 30s at 60°C, 40s at 72°C10 min at 72°Chold at 4°C
?. [Library amplification]
Verify the reactions using agarose gel electrophoresis.
?. [Library amplification]
Pool the samples, including the 'blank' samples.
?. [Library amplification]
Purify the pool using standard AMPure XP protocol using 0.8x ratio of AMPure and elute in 20 uL of nuclease-free water or 10mM Tris.
?. [Size selection]
Perform size selection using PippinPrep instrument or by cutting the desired fragment from the gel.
?. [Final quantification]
Check size selection and library fragment size using gel electrophoresis or TapeStation/Bioanalyzer/Fragment Analyzer.
?. [Final quantification]
Quantify the amount of DNA using Qubit.
?. [Sequencing]
Proceed to HiSeq/MiSeq sequencing. DNA quantity and mean fragment length can be used to calculate the molarity of the library.
TITLE: Multiple Sequence Alignment
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Multiple Sequence Alignment

[STEPS]
1. Protein sequences were retrieved from Uniprot or NCBI via Jalview’s sequence fetcher Jalview
(RRID:SCR_006459)(https://www.jalview.org/help/html/features/seqfetch.html).







2. Then, the sequences were aligned with the
Clustal Omega Webserver [(RRID:SCR_001591) http://www.ebi.ac.uk/Tools/msa/clustalo/]
with default parameters. 
3. For the α-synuclein alignment human (uniprot OP37840), chimpanzee (uniprot P61145), baboons
(uniprot  A0A2I3N0Z9), marmoset (uniprot F7GY62), rat (uniprot P37377), mouse (uniprot O55042), rabbit (uniprot G1U0V2),
cow (uniprot Q3T0G8), bat (uniprot A0A6J2L5S3), shrew (uniprot J7H0X3),
armadillo (NCBI XP_004480597), elephant (uniprot G3T7Z3), tenrec (NCBI
XP_004703317.1), chicken (uniprot Q9I9H1), zebra finch (uniprot Q4JHT6), frog
(uniprot Q7SZ02),  and fish (uniprot
A0A090D865) sequences were used.
TITLE: Latest Pakistan News
AUTHORS: Tamara  Walsky, Ariana  Macon

[DESCRIPTION]
Please provide an abstract for your protocol before publication.

[STEPS]
1. Latest Pakistan News Na­tion cel­e­brates vic­to­ry in 1971 in­de­pen­dence war by host­ing  mil­i­tary pa­rade with In­dia’s pres­i­dent as visitor of ho­n­our.

 Qatil Haseenaon Ke Naam has femme fa­tales in saris, jhum­ka ear­rings  as Professional­tag­o­nists and may be streamed in one hundred ninety  na­tions.

 Officials have described the supervisor of the factory, who was a Sri  Lankan, was killed as locals thought that he had committed blasphemy

 Pro­check­ers march to­wards shut­tered US em­bassy, phone­ing for  re­lease of Afghan as­sets, frozen given that Tal­iban took pow­er.

 Apr 14, 2013· Know the very best about 10 items to import from Pakistan:  Pakistan is an average financial state country, exactly where lots of  sorts of points are produced. All shortlist of those issues is prepared  over, we hope you will just like the stuff but keep in contact with  since There exists coming more.

 Siachen is noted for tragedies, a desolate spot exactly where additional  troops have died from avalanches or bitter chilly than in fight. Due to  the fact getting independence from Britain in 1947, Pakistan and India  have fought two in their three wars over Kashmir.Read more

 We also use 3rd-party cookies that assistance us review and understand  how you use this Web page. These cookies is going to be saved within  your browser only with the consent. You also have the option to  choose-out of these cookies. But opting from Some cookies might impact  your browsing practical experience.

 “Key Minister Imran Khan has convened a gathering in the Countrywide  Security Council tomorrow, during which the very first countrywide  safety coverage while in the background from the state are going to be  introduced for acceptance,” the Pak minister had mentioned in a tweet.Pak news

 When govt difficulties any highly effective phase of society for just  about any illegality, they start threatening to topple it, claims AGP.

 Having the difficult highway could assure India gains a far more highly effective Breaking News from All over Pakistan position from the post-pandemic international stage.

 The J-10C aircraft have been Portion of the Pak-China joint exercising  previous 12 months, wherever industry experts from Pakistan experienced  the opportunity to have a detailed look at these.

 The mil­i­tary stated on Fri­day that se­cu­ri­ty forces raid­ed two  conceal­outs inside a for­mer Tal­iban potent­maintain in close  proximity to Afghanistan.

 Formerly the ultimate source of getting news was only by means of  newspapers and that also within their printed from. Now all which has  modified as individuals now not depend upon just printed newspapers to  remain updated. During this fashionable globe, the fast resources of  having news are TV news channels, digital newspapers, radio, and social  media marketing.

 Finance supplementary Monthly bill to become debated; Speaker lets extension in lifetime of 6 expired ordinances.
2.
TITLE: Masson-Fontana Staining
AUTHORS: Nuriapenuelas

[DESCRIPTION]
Masson-Fontana Staining for melanin detection

[STEPS]
1. Heat paraffin tissue sections at 60ºC for 10min.
2. Incubate sections in xylene (5 min, 3 times) and in ethanol serial washes (100-95-70%-H2O, 5 min each). 
4. Incubate the sections with sodium thiosulphate for 2 min. Rinse sections in distilled water.
3.                 
Incubate the sections with ammoniac solution for 40 min at 56ºC. Rinse sections in distilled water.

5. Finally, counterstain with Kernechtrot for 7 min. Rinse sections in distilled water.
TITLE: Cryptococcus neoformans DNA Extraction Method
AUTHORS: Amy Gladfelter

[DESCRIPTION]
Extraction method to obtain genomic DNA from Cryptococcus neoformans.

[GUIDELINES]
Extraction buffer (100 ml) 


 

CTAB is mixed alkyltrimethyl ammonium bromide, Sigma cat.# M7635. This takes time to go into solution. You can also use solid NaCl rather than a 5 M solution if that is easier. The buffer lasts four- six months at room temperature.The buffer seems to work better for long strands and spooling if β-mercaptoethanol is added just prior to use. 
 
 
 
 
 
 
 
  

[STEPS]
1. Grow a 50 mL YPD culture overnight (960 min), shaking at 30 °C.



2. Pellet cells in tabletop centrifuge in a 50 ml disposable tube.
 

3. Freeze cells at -20 °Cto -80 °C for <30 min, then dry in a freeze drying machine.


4. Add the equivalent of 3 mL to 5 mLof 2 mm glass beads and vortex/shake until the cell pellet is broken and a fine powder is created.


5. In fume hood, add 10 mL CTAB extraction buffer (see Guidelines) and mix.  

7. In fume hood, add 10 mL chloroform and gently mix for approximately 1 min.


8. Spin in a table top centrifuge for 10 min (2,500 – 3,000 rpm).

9. Remove supernatant (c. 7 ml) and add to an equal volume of isopropanol in a 15 ml disposable tube.
10. Gently rock back and forward to mix.

If the DNA precipitates in strands and clumps, spool out with a glass pipette and transfer to eppendorf containing 1 mL70% ethanol. 

Otherwise, spin in a table top centrifuge for 10 min, pour off supernatant and use 1 mL 70% ethanol to wash DNA pellet and transfer it to an eppendorf tube.




11. Spin sample in microcentrifuge for 5-10 minutes. Remove ethanol and allow to air dry.


12. Resuspend DNA in either water or TE buffer (c. 500 μl). 

RNase can be added to final concentration of 20 μg/ml if needed. 

Run 1 µL on an agarose gel to check concentration and quality.

6.  Incubate at65 °C for 30 min.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This year´s interlab study allows participants to fulfil Bronze medal requirements. </p>
<p>For this purposes a set of experiments has to be performed, which in the end will be compared and validated with other team´s data. </p>
<p>Part of this Challenge are the Calibration protcols. </p>
<p>For the second calibration a dilution series of monodisperse microspheres is performed and absorbance at 600nm is measured. </p>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<p>Make sure to adjust the temperature of your plate reader to room temperature (22°C-25°C) before measurement.</p>
</div>
</div>
</div>
</div>
</div>
<div> </div>
<div>Also prepare the silica beads stock solution before starting. Follow the steps in this protocol. </div>

[GUIDELINES]
<p>Make sure to always use the same plates, volumes and settings for the measurement as will be used for the other calibrations and the cell measurements. </p>
<p>Also take care to remain constant with your pipetting techniques. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Cellular lipid uptake with flow cytometry readout
AUTHORS: Igor Beletchi, rosanne.wouters, Peter Vangheluwe

[DESCRIPTION]
Cellular lipid uptake with flow cytometry readout

[STEPS]
SECTION: harvest cells
1. harvest cells by detachment with  

SECTION: harvest cells
1.1. cells should be grown to 70% confluency, not higher
SECTION: harvest cells
2. collect cells by centrifugation 400 x g, 5 min, 4 °C 

SECTION: harvest cells
3. wash pellet with with  
SECTION: harvest cells
4. collect cells by centrifugation 400 x g, 5 min, 4 °C 
SECTION: harvest cells
5. resuspend pellet  in   
SECTION: harvest cells
6. count cells
SECTION: harvest cells
7. prepare cell suspension with 1*10^6 cells in 500µl   

SECTION: prepare lipid suspension
9. wash glass syringe 3x with 100% ethanol and 3x with chloroform, let dry
SECTION: harvest cells
1.2. wash cells with  

SECTION: harvest cells
1.3. incubate cells with  (3ml/T175 flask) at room temperature

SECTION: harvest cells
1.4. when cells detach resuspend with 7ml    and keep on ice in 15 mL falcon

SECTION: lipid uptake
13. equilibrate cell suspension   
SECTION: lipid uptake
14. equilibrate lipid suspension    
SECTION: prepare lipid suspension
8. work under fume hood equipped with N2 flow
SECTION: prepare lipid suspension
10. with glass syringe pipette needed volume of NBD-lipid into glass vial   ,  ,  ,  ,   ,  

SECTION: prepare lipid suspension
10.1. keep glass bottle with NBD-labeled lipids in chloroform solution cold, by working on ice or in freezer block
SECTION: prepare lipid suspension
11. dry lipids by evaporating chloroform under N2 flow
SECTION: prepare lipid suspension
12. resuspend lipids in   to final concentration of 2µM with magnetic stirbar until fully dissolved, keep lipids at 4°C while dissolving

SECTION: lipid uptake
15. prepare collection tubes with 200µl  containing 5%    (per sample 1 collection tube is needed for each timepoint) and keep on ice

SECTION: lipid uptake
16. for timepoint 0, take 200µl of equilibrated cell suspension and add to collection tube T=0 (containing 200µl of ice-cold   containing 5%   ) on ice
SECTION: lipid uptake
17. add 500µl of lipid suspension to remaining 300µl of cell suspension
SECTION: lipid uptake
18. incubate at 37°C with  thermoshaker with interval 1 min shaking every 5 min (700 rpm).
SECTION: lipid uptake
18.1.  keep samples away from light during incubation
SECTION: lipid uptake
19. after 30min, take 200µl of lipid+cell suspension and add to collection tubes T=30 (containing 200µl of ice-cold   containing 5%   ), keep on ice
SECTION: lipid uptake
20.  after 60min, take 200µl of lipid+cell suspension and add to collection tubes T=60 (containing 200µl of ice-cold  containing 5%   ), keep on ice
SECTION: lipid uptake
21. add sodium dithionite to all samples (1/100 dilution of freshly prepared 1M stock in tris-buffer, pH10) and vortex samples (make sure to mix well with quencher)
SECTION: lipid uptake
22. measure total internal fluorescence with flow cytometer
SECTION: prepare lipid suspension
10.2. for each sample 500µl lipid suspension with 2µM final concentration is needed
TITLE:      ELISA for quantification of IL-27 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>T cells make IL-27 that activates STAT-1 and STAT-3, which regulates immune responses. IL-27 stimulates IL-10 production. It is a pro-inflammatory molecule and upregulates type-2 interferon synthesis by natural killer cells. </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-27 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-27 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-27 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-27 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-27 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-27 standard dilutions and the human IL-27 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.