eamag/protocols-with-steps huggingface dataset:
TITLE: Isolation and culture of primary mouse brain microvascular endothelial cells
AUTHORS: barbara

[DESCRIPTION]
Immunofluorescent characterization of BMECs. The isolated BMECs were 95% positive for CD31(red), confirming the epithelial nature of these cells. 


[STEPS]
1. 
2. Isolation and culture of primary mouse brain microvascular endothelial cells

Mouse brain microvascular endothelial cells (BMECs) were prepared from 2-week-old C57BL/6 mice as previously recommended [81]. Mice was soaked in 75% ethanol to disinfect 5 min after the cervical dislocation, then mice brains were removed and placed in a 10 cm petri dish containing precooled Hank’s solution. The white matter, residual blood vessels and pia mater were removed and the cerebral cortex was retained. Brain tissue was minced and homogenized, and 10 ml Modified Eagle Medium (MEM) medium was added to the tissue suspension and centrifuge the cells at 1200 rpm for 10 min at 4°C. The supernatant was discarded and myelin was removed by centrifugation at 2600 r/min for 10 min in 22% bovine serum albumin (BSA, Sigma). The microvascular segments and cells were resuspended by using the Endothelial Cell Growth Media (ECGM) and centrifuge at 1200 r/min for 5 min. Then the BMECs were inoculated in 12-well culture dishes previously coated with type I collagen. BMECs were cultured in 5% CO2 incubator at 37℃ with medium containing 80% Dulbecco’s Modified Eagle Medium (Gibco, cat# 10566016), 15% fatal bovine serum (Gibco, cat# 16010142), 0.05% endothelial cell growth supplements (Millipore, cat# 211F-GS), 0.1% heparin (Sigma, cat# H9267) and 1% antibiotic-antimycotic solution (100X, Invitrogen, cat# 15240096). On the second day, endothelial cell growth medium (ECGM) containing 4 μg/mL puromycin (Sigma, cat# P8833) was added to continue culture, and 72 h later it was replaced with conventional ECGM medium. The culture medium was changed every other day, and when the cells reached 90-95% fusion, 0.25% trypsin was used for passage. BMECs were seeded at a density of 1.0 × 104 cells/cm2 for subsequent experiments and passaged when BMECs reached 90% confluence. Serial passage of cells was performed by trypsinization with trypsin-EDTA (Gibco, cat# 25200072). The purity of BMECs was controlled by immunofluorescence staining of endothelium marker CD31 (1:500, CST, USA, cat# 3528S).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The kit is designed for the isolation of human memory CD8 T cells from PBMC. Target cells are depleted by incubating the sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>
<p> </p>

[GUIDELINES]
<p>MojoSort™ magnetic particles can be used with other commercially available magnetic separators, both free standing magnets and column-based systems. Because MojoSort™ protocols are optimized for the MojoSort™ separator; the protocols may need to be adjusted for other systems. Please contact BioLegend Technical Service (tech@biolegend.com) for more information and guidance. We do not recommend using MojoSort™ particles for BD’s IMag™ or Life Technologies’ DynaMag™.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Each P-RANbody consists of a HRP reporter and a nanobody that binds to antigen(s). This is a protocol to stain cells and tissues with P-RANbodies. The method is similar to common immunostaining protocols.  However, this protocol focuses on some important tips.  </p>
<p> </p>
<div title="Page 2">
<p> </p>
</div>

[BEFORE_START]
<p>P-RANbody plasmids can be obtained from Addgene. </p>
<p><a href="https://www.addgene.org/browse/article/28192638/" target="_blank" rel="noopener noreferrer">https://www.addgene.org/browse/article/28192638/</a></p>

[GUIDELINES]
<p>The method is similar to common immunostaining protocols, but requires some knowledge. </p>
<p>1) Endogenous peroxidase activity can be found in various cells and tissues.  It is critical to inactivate this activity before incubating with P-RANbodies. </p>
<p>2) P-RANbodies contain HRP.  HRP is sensitive to sodium azide, a common preservative. </p>
<p>3) TSA is a reagent which often requires extensive washing after reaction.  </p>
<p> </p>
<p>For questions or discussion, please post them on the RANbody forum. </p>
<p><a href="https://www.protocols.io/groups/ranbody" target="_blank" rel="noopener noreferrer">https://www.protocols.io/groups/ranbody</a></p>

[STEPS]
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?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For <a href="https://www.protocols.io/view/Transcriptomics-During-One-Step-Growth-Curves-for-dem3c5" target="_blank">Transcriptomics During One-Step Growth Curves for Cellulophaga Phages protocol</a>.

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: How to feed Nellie and Mira (or any two cats)
AUTHORS: Gabriel Rocha

[DESCRIPTION]
My cats Nellie and Mira have developed an annoying habit of wanting to eat different amount of food, at different times. This is the best way to make sure they both eat everything in one go so I don't have to worry about one of them eating the other's food.

[STEPS]
SECTION: Preparing the food mat
1. Get food mats likely on the floor of the living room and clean leftover food with soap and a scrub
SECTION: Preparing the food mat
2. Dry the silicone mat, especially the underside to prevent mould growth
SECTION: Preparing the food
3. Grab wet food can from cabinet above fridge and open the container
SECTION: Preparing the food
4. Split the contents in half, making sure each food mat gets 35 g  of wet food

SECTION: Preparing the food
6. Add 20 g of dried kibble located to the left of the oven to each mat next to the wet food

SECTION: Feeding Nellie and Mira
7. Bring one mat to Mira, who will immediately lunge towards her food
SECTION: Feeding Nellie and Mira
8. Grab the other mat and bring it to a different room so Nellie can eat in peace and not be bullied by Mira to eat more
SECTION: Feeding Nellie and Mira
9. Stay with nellie for 5 min  so she feels safe while eating

SECTION: Finish
10. Finish by cuddling both of them and telling them how pretty they are


 





SECTION: Preparing the food
5.1. Nellie is a picky eater, so add a few bites of the mackerel treat stored in fridge 4 °C  to increase her appetite
SECTION: Preparing the food
5. Spread the wet food through the pad so it has a thickness of about
TITLE: Purification and analysis of SKP1-FBXO7 complexes
AUTHORS: Frank Adolf, Brenda A. Schulman

[DESCRIPTION]
Protocol for the biochemical purification and analysis of SKP1-FBXO7 complexes

[GUIDELINES]
Please wear appropriate PE while performing the experiment.
Please familiarise yourself with the laboratory safety rules and guidelines and follow these while performing the experiment.

[STEPS]
SECTION: Molecular biological methods and protein expression
2. Grow E.coli cultures in Terrific Broth (TB) medium at 37°C.
At OD600 of 0.8, induce expression with 0.5mM IPTG.
Continue growing the E.coli culture for 16h at 18°C.
SECTION: Analytical size exclusion chromatography (SEC)
7. Analytical SEC was carried out on an ÄKTApure system (GE Healthcare) equipped with a Superdex 200 Increase 10/300 GL column (Cytiva), in buffer A (25mM HEPES pH7.5 (KOH), 150 mM NaCl, 1mM DTT). 
SECTION: Preparatory note
1. All constructs were prepared utilizing standard molecular biological techniques and verified by sanger sequencing. 

The cDNAs coding for HsFbxo7-129-398 and Skp1 preceding a second RBS were cloned into pGEX4T1 as previously described for other substrate receptor/Skp1 complexes (Schulman et al. 2000) (pGEX4T1-TEV-HsFbxo7-129-398/HsSkp1). 

The cDNA coding for HsPI31-1-151 (with N-terminal TEV cleavable His8-tag) was cloned into pRSF1b (pRSF1b-His8-TEV-HsPI31-1-151). 

For co-expression of Fbxo7/Skp1/PI31 complexes, both plasmids were co-transformed into E. coli BL21 Rosetta (DE3).

SECTION: Molecular biological methods and protein expression
5. Pool fractions of interest, aliquoted and snap freeze in liquid N2.
Store fractions at -80°C until further usage. 
SECTION: Molecular biological methods and protein expression
6. HsCul1-1-410 was expressed as GST-fusion protein and purified as described previously (Hopf et al., 2022).
SECTION: Molecular biological methods and protein expression
3. Purify FBXO7/Skp1/PI31 complexes by sequential standard GST- and His-affinity chromatography.
Cleave affinity tags by incubation with TEV protease at 4°C for 16h.
SECTION: Molecular biological methods and protein expression
4. Further purify complexes by preparative size exclusion chromatography (SEC) in buffer A (25mM HEPES pH7.5 (KOH), 150 mM NaCl, 1mM DTT; for details please see next section) on a Superdex 200 Increase 10/300 GL column.
SECTION: Analytical size exclusion chromatography (SEC)
9. Apply samples (100 µl at a concentration of 45 µM) on column.
Set flow rate to 1 ml/min.

SECTION: Analytical size exclusion chromatography (SEC)
8. Preincubate samples at 37°C for 10 min before loading.
Samples:
HsCul1-1-410, 
HsFbxo7-129-398/HsSkp1/HsPI31-1-151
HsFbxo7-129-398/HsSkp1/HsPI31-1-151 + HsCul1-410
SECTION: Analytical size exclusion chromatography (SEC)
10. Record UV absorbance at 280 nm and collect fractions of 200 µl volume.

SECTION: Analytical size exclusion chromatography (SEC)
11. Analyze fractions by SDS-PAGE.
TITLE: A method for the permeabilization of live Drosophila melanogaster larvae to small molecules and cryoprotectants
AUTHORS: Alex Murray, Daniel Palmer, Daimark Bennett, Venkata Dwarampudi, João Pedro de Magalhães1

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The larvae of </span><span style = "font-style:italic;">Drosophila melanogaster </span><span>is a model organism used to study the muscular and nervous systems. </span><span style = "font-style:italic;">Drosophila</span><span> larvae are surrounded by a waxy cuticle that prevents permeation by most substances. Here we develop a method to remove this layer, rendering the larvae permeable to small molecules. Permeability is assessed using fluorescein diacetate dye uptake, and mortality upon exposure to toxic levels of ethylene glycol and DMSO. Potential uses for this method include drug delivery, toxicity assays, cryopreservation, staining, and fixation. </span></div></div>

[STEPS]
TITLE: Protocol 2: Culturing Spizellomyces punctatus (Sp) prior to transformation day
AUTHORS: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

[DESCRIPTION]
In order to increase transformation efficiency, a more synchronous culture of Sp is needed. This process involves subculturing active Sp plates in the days leading up to transformation day. At least two generations of Sp should be grown on antibiotic free K1 medium to ensure no trace antibiotics are present in the culture for transformation day. Proper sterile technique should be followed at all times when maintaining cultures of Sp; use either a laminar flow hood or work in the sterile area around an open flame.

[BEFORE_START]
In order to prepare enough plates to provide an adequate amount of zoospores for transformation, subculture one plate of Sp per plasmid to be transformed. One active plate is enough to subculture 2-3 new plates. Complete this protocol roughly 36 hours and then again roughly 18 hours prior to the intended transformation time.

[STEPS]
SECTION: Steps
1. Flood enough active Sp plates each with 1 mL of DS to fit your needs. 
 

SECTION: Steps
2. Holding the plate at an angle, gently wash the DS over the agar.
SECTION: Steps
3. Collect all zoospores into a 50 mL conical.
SECTION: Steps
4. Filter all zoospores into a new 50 mL conical using the sterile 25 mm syringe filter preloaded with Whatman Grade 1 filter paper.
SECTION: Steps
5. Inoculate the proper amount of new K1 plates for your needs with 250 µL to 500 µL of filtered zoospores. 
 



SECTION: Steps
6. Add 500 µL of fresh DS to the newly inoculated plates.

SECTION: Steps
7. Gently tilt the plates to spread the liquid across the entire surface of the agar.
SECTION: Steps
8. Incubate at 30 °C in a humidity chamber. 
 

SECTION: Steps
2.1. Wash the 1 mL of DS over the agar. (1/3)
SECTION: Steps
2.2. wash the 1 mL of DS over the agar. (2/3)
SECTION: Steps
2.3. Wash the 1 mL of DS over the agar. (3/3)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to make an egg dechorionator using available lab equipment before describing how to successfully use the egg dechorionator to remove the outer chorion layer from a Drosophila egg. 

Egg dechorionation allows cellular analysis, quantitative microinjection and the rearing of developing offspring in a gnotobiotic or axenic environment. 

This protocol is written with the use of dechorionated eggs for rearing fruit flies in a gnotobiotic or axenic environment in mind. Therefore sterile techniques are described. For cellular analysis and/or quantitative microinjection the protocol can be followed but with a less stringent sterile procedure where unneccesary.

[GUIDELINES]
Gloves should be worn during all steps of the protocol. 
Step 1 should be performed in a fume hood. 
Step 7-onwards should be performed in a laminar air flow hood with sterile conditions. The flow hood and all equipment added to the hood must be cleaned thoroughly with 70% ethanol before commencing.

Following these guidelines will reduce the possible contamination of your eggs with any microorganisims.

[STEPS]
SECTION: Making the egg dechorionator
?. 
SECTION: Preparing apple agar
?. 
SECTION: Setting up the egg cage
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?. 
SECTION: Egg dechorionation
?.
TITLE: SPARC Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons Using Intrapleural Injections of Cholera Toxin B Fragment
AUTHORS: Latoya Allen, Marissa Ciesla, Yasin Seven, Elisa Gonzalez-Rothi, Gordon Pool

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for retrogradely labeling bilateral phrenic motor neuron pools in the cervical spinal cord using intrapleural injections of cholera toxin B fragment</div></div>

[STEPS]
?. Anesthesia is induced in a closed chamber (3.5%isoflurane in 100%O2) then transfered to a heated surgical station and anesthesia is maintained via nose cone at 2-2.5%
?. Sterilized 25 μL Hamilton syringes with 9.52-mm sterile needles are prepared to administer CtB intrapleurally in rats.
?. Cholera toxin B fragment (CtB) is prepared as follows: 0.2% weight/volume; dissolved in sterile H2O; Calbiochem, Billerica, MA.
?. 25 μL of CtB is loaded into a 25-μL Hamilton syringe and 12.5 μL is  injected per side (25μL total per animal).
?. Prep injection site for injection using aseptic methods:
?. shave and scrub injection sites per Institutionally approved animal protocol
?. Rats were turned sideways to inject CtB at the 5th intercostal space at a depth of ~6 mm.
?. Following injections, discontinue isoflurane and allow rat to recover fully while monitoring for signs of respiratory compromise or distress
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span lang="en"><span class="">In this protocol, the extraction of naturally occurring as well as heterologously synthesized triterpenes in <em>Synechocystis </em>and <em>R. capsulatus</em>  are described</span></span></p>

[BEFORE_START]
<p>Prepare high-quality solvents (opt. LC-MS grade) and add 0.05% BHT (butylated hydroxytoluene)  to all solvents (acetone, n-hexan, chloroform/methanol [2:1])</p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: Ruegeria pomeroyi DSS-3 gene annotations - April 2020
AUTHORS: Christa Smith, Mary Ann Moran

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Ruegeria pomeroyi</span><span>  DSS-3 is an alphaproteobacterium isolated in 1998 from coastal Georgia, USA, seawater by enriching for growth on the organic sulfur compound dimethylsulfoniopropionate (DMSP) (González and Moran 1997, Buchan et al. 2001).</span></div><div class = "text-block"><span style = "font-style:italic;">R. pomeroyi </span><span>is a member of the Roseobacter group, which consists of marine bacteria within the family Rhodobacteraceae that are important members of bacterioplankton communities in coastal and open oceans. </span><span style = "font-style:italic;">R. pomeroyi</span><span>  grows quickly in the laboratory and has a genetic system. Since its genome sequence became available in 2004 (Moran et al., 2004), </span><span style = "font-style:italic;">R. pomeroyi</span><span>  has been serving as a model organism for studies of the biogeochemical,  ecological, and physiological strategies of heterotrophic marine bacteria.</span></div><div class = "text-block"><span>This document catalogs the current manually curated gene annotations of the </span><span style = "font-style:italic;">Ruegeria pomeroyi </span><span>DSS-3 genome, updated as of April, 2020.</span></div></div>

[STEPS]
TITLE: Cell growth and harvest for E. coli-based Cell-free Protein Synthesis
AUTHORS: Weston Kightlinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cell-free protein synthesis reactions require cell-free extract derived from rapidly dividing E. coli cells to produce high yields of protein therapeutics, antigens, enzymes, vaccines, and other proteins. This protocol describes the cell growth and harvest steps for E. coli to produce extract.</div><div class = "text-block"><span>Read more on cell-free protein synthesis here: </span><a href="https://www.swiftscalebio.com/blog/cell-free-protein-synthesis" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.swiftscalebio.com/blog/cell-free-protein-synthesis</span></a><span style = ":;"> </span></div></div>

[STEPS]
?. [Cell growth and harvest for E. coli-based Cell-free Protein Synthesis]
Start a  seed culture of E. coli strain and grow in LB media  at .
50 mL
37 °C
?. [Cell growth and harvest for E. coli-based Cell-free Protein Synthesis]
Add  glucose to  2xYTP media in Tunair flask to obtain 2xYTPG media.
250 mL
750 mL
?. [Cell growth and harvest for E. coli-based Cell-free Protein Synthesis]
Inoculate Tunair flask culture at approximately OD600 = 0.05.
?. [Cell growth and harvest for E. coli-based Cell-free Protein Synthesis]
Grow culture at  with shaking at  to OD600 = 0.6 and induce T7 with IPTG (if necessary). Add to  IPTG.
37 °C
Centrifuge: 250 33
?. [Cell growth and harvest for E. coli-based Cell-free Protein Synthesis]
Harvest culture at OD600 = 3 by centrifugation at  for  and washing with S30 buffer three times.
Centrifuge: 8000 34
?. [Cell growth and harvest for E. coli-based Cell-free Protein Synthesis]
After the last wash, pour off S30 supernatant to obtain the pellet. Resuspend this pellet in S30 and lyse or immediately flash freeze on liquid nitrogen and store at  until ready for cell lysis.
-80 °C
TITLE: Cup of tea (polish way)
AUTHORS: tomasz.zielinski , ines.boehm 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is part of the lesson within the </span><a href="https://carpentries-incubator.github.io/fair-bio-practice/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">FAIR in (biological) practice</span></a><span> Carpentry course. Here we explore the perfect protocol to brew tea the polish way.</span></div><div class = "text-block">Tea in Poland is served in transparent glasses to admire the colour of the tea and judge its "strength". Tea is never served with milk but often with a bit of lemon.</div></div>

[STEPS]
?. [Preparation of water and tea]
Fill the kettle with over 250mL of water, or until your min fill line and boil the water.
?. [Preparation of water and tea]
In the meantime prepare your tea bag, unpack it and place it in the transparent glass (or a mug). If the paper tag is present, position it outside of the mug.
?. [Preparation of water and tea]
Once the water is boiled, instantly pour the hot water into the mug. Hint: Ensure you fix the tag on the outside of the mug with your finger, to avoid falling in of the string + tag of the tea bag. Otherwise removal of the tea bag becomes more difficult.
?. [Brewing]
Brew the tea for 1 to 3 min, keep an eye on the changing color you want to interrupt brewing once it reaches light brown, like oak wood. Don't wait too long till it becomes dark brown that is considered a too strong tea in Poland.
?. [Brewing]
Use the string/tag to remove the tea bag. Discard the tea bag in your compost for most appropriate recycling.
?. [Flavouring]
Add 2 teaspoons of sugar
?. [Flavouring]
Enjoy!
?. [Flavouring]
Wait 5 minutes and either squizz in few drops of lemon or insert a lemon slice. See how the color changes to bright brown or yellow. Hint: Never add lemon to boiling hot tea as it destroys the natural vitamin C.
TITLE: Cas9 RNP nucleofection for CD34+ HSPCs using Lonza 4D Nucleofector
AUTHORS: Mark DeWitt

[STEPS]
?. 
?. [Prepare RNP mix]
?. Add Cas9 to sgRNA slowly while swirling pipette tip, should take 30s to 1 minute.
?. 
?. Count cells using hemacytometer.
?. For each nucleofection, pipette 200k cells into a 15 mL conical.
For replicates, you can multiply the amount of cells and RNP mix as needed and mix in a single tube before electroporating separately.
?. [Prepare Cells]
Spin 200 x g for 10 minutes to pellet cells softly. While the cells are spinning, prepare plate and cuvette.
?. [Prepare Cells]
Place a 96 well plate in the tissue culture incubator.
?. [Nucleofection]
Prepare and label wells on 20uL nucleofection strips. Configure Lonza 4d to zap with code ER100.
?. [Nucleofection]
Pipette off media from cells, gently but completely, using a P200. The pellet is very soft so be careful.
?. [Nucleofection]
Resuspend cells in 20 µL of P3 nucleofector solution using a P200.
?. [Nucleofection]
Add the entire 10 µL RNP mix to the 20 µL resuspension and mix.
?. [Nucleofection]
Add 1uL of 100uM donor DNA (100 pmoles) and mix well.
?. [Nucleofection]
Add 27.5 µL of cells/RNP electroporation mix to the multiwell cuvette, and cap. Pay attention to the orientation of the cap and cuvette in the nucleofector, which is noted in the manufacturer’s instructions.
?. [Nucleofection]
Insert cuvette into nucleofector and zap.
?. Immediately layer electroporated cells with 72.5 µL of medium (SFEM II/Pen-strep/CC110) and rest for 5 minutes at room temperature.
?. 
?. [Nucleofection]
Pipette mixture out with a P200 into your pre-warmed 96 well plate. Wash each cuvette with an additional 100 µL of medium, and add that as well (final plating volume 200 µL).
?. [Nucleofection]
Culture cells for several days in SFEM II/PenStrep/CC110 before genotying by desired method. Consider including un-zapped controls to test viability.Alternatively, transfer cells to expansion medium after 1-2 days. We have used StemCell Erythroid Expansion supplement for this purpose. Otherwise, the cells will not expand substantially if left in CC110
?. [Thaw HSPCs, plate at 300,000 cells/mL in stem cell expansion medium]
?. [Count cells daily using hemacytometer]
Log counts for day 0 (thaw day), day 1, and day 2 (electroporation day).Generally, the count will decline slightly from day 0 to day 1, and recover from day 1 to day 2. The total number of cells on day 2 will be slightly higher than on day 0.