eamag/protocols-with-steps huggingface dataset:
TITLE: Thawing, Passaging and Freezing of hPSCs on MEFs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).

Collection overview
Thawing of hPSCs grown on MEFs
Passaging of hPSCs grown on MEFs
Freezing of hPSCs grown on MEFs
  A. Freezing of hPSCs as single cell suspension using trypsin 
  B. Freezing of hPSCs as cell aggregates using collagenase 

General notes
1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions.
3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.
4. While bulk/collagenase passaging is used for routine maintenance of hPSC cultures,
manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires (i) the identification and discrimination of undifferentiated and differentiated hPSC colonies and (ii) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.

[STEPS]
TITLE: U Mass -Lactate
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.  Serum lactate levels are affected by alterations in glucose and protein metabolism.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Lactate test on display and run the analysis.
?. Collect and analyze the data.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol has been optimized to remove washing steps after antibody cocktail and nanobeads incubations, resulting in a shorter and more convenient protocol. This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube from human peripheral blood mononuclear cells (PBMCs). If working with fewer cells keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. To maximize the purity of CD4+ T cells we recommend the depletion of monocytes, as human monocytes express CD4. To deplete monocytes from human PBMCs, we recommend the use of MojoSort™ Human CD14 Selection Kit (Cat. No. 480025/480026). Alternatively, you may consider the Human CD4 T Cell Selection Kit (Cat. No. 480038). After monocyte depletion proceed to isolate the CD4+ T cells. You can also use other methods to deplete monocytes. If you wish to include monocytes in your CD4+ cell isolation, depletion is not required</p>

[GUIDELINES]
<p><strong>Product description and procedure summary:<br /></strong>The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>
<p> </p>
<p><strong>Reagents and instruments required:</strong></p>
<p><br />MojoSort™ Buffer (5X) (Cat. No. 480017)<br />MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system<br />Adjustable pipettes<br />70 μm filters (one per sample)<br />5 mL (12 x 75 mm) polystyrene tubes<br />Reagents for sample preparation<br />Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dsbecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dsaecje.jpg" data-ofn="mojosort human cd4 no wash.JPG" /></p>
<p><strong>Application notes:</strong> To use this product in magnetic separation columns, a titration of the Nanobeads should be performed. Optimal concentration for magnetic separation columns is lot-specific.</p>

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TITLE: Determination of IgM concentration by the Mancini test.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. An appropriate anti-IgM antiserum (antibody) is poured in the center well of an agar-containing plate.
?. Carefully circular wells are cut and detached from the plates.
?. A series of standards containing known concentrations of IgM are placed in separate wells, while “unknown” human serum samples and control are placed in other wells.
?. A ring of precipitate forms in the area of optimal antigen-antibody concentration, meaning anti-IgM - IgM reaction as the antigen diffuses radially.
?. The diameters of the rings are measured and perceived normally in 48-72 hours.
?. Finally, a standard curve is developed using the ring diameters of the standards versus the concentrations.
?. A curve is then used to plot the concentration of the control and unknown IgM samples.
TITLE: Isolation of Nuclei from Adult_Human_Brain_Tissue
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Isolation of nuclei from frozen adult human brain tissue or thawed and microdissected brain tissue sections for FPCR and/or RNA-seq analysis. </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: CD34+ isolation from human bone marrow
AUTHORS: Mohsen Khosravi-Maharlooei, Markus Holzl, Austin Chen, Megan Sykes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the steps for isolating CD34+ cells from human bone marrow. The CD34+ cells isolated from this protocol can be used for generating humanized mice through reconstitution of immune cells via IV injection after bone marrow ablation. These cells can also be used for mixed lymphocyte reaction experiments.</div><br/><br/><br/><br/></div>

[STEPS]
?. Transfer the content of the collection bag into a sterile flask
?. Add BM Medium to the bag and rinse it thoroughly
250 mL
?. Transfer the content of the collection bag into the sterile flask
?. Layer of the suspension over  of Histopaque
35 mL
15 mL
?. Centrifuge the tubes for 30 minutes  without brake at RT
500 g
?. Collect the leukocyte ring in  Falcons and fill up with BM medium
50 mL
?. Wash once by centrifuging 6 minutes
500 g
?. Resuspend the cells in MACS buffer and count (take for FACS confirmation = PRE)
50 µl
?. Wash down again and resuspend the pellet according to the protocol (130-046-702 MACS Human CD34+ kit)
?. Add  of MACS buffer per 10^8 cells
300 µl
?. Add  of FcR-B reagent per 10^8 cells, mix it and incubate in fridge for 15 minutes
100 µl
?. Add of CD34 beads per 10^8 cells to the suspension and mix and let it sit for 30 min (fridge)
100 µl
?. Fill up with  MACS buffer and strain through a blue strainer (40 uM)
50 mL
?. Wash cells (6 min) and resuspend in MACS buffer 500 uL/200,000,000 cells. If you have more cells, increase volume accordingly. E.g 3 *10^9 cells = 7,5mL. Aliquot this volume to more than one (with  prerinsed) MACS column.
500 g
3 mL
?. Wash with buffer  3 times and keep negative fraction (Take  for FCM = POST neg)
3 mL
50 µl
?. Put the column out of the magnet and push out positive fraction with  Buffer and the plunger
5 mL
?. Collect the positive fractions. (Take  for FCM = Post pos)
50 µl
?. Process the cells as desired (injection in mouse or cryopreservation)
?. Check the puritiy with FACS
?.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
** It is IMPORTANT to use a buffer that matches what the virus is resuspended in as it helps keep the virus heads intact.&nbsp; If you are looking at a CsCl gradient fraction that has not been dialyzed, we find that as the diluent using CsCl with a similar density helps keep the viruses intact.&nbsp; If the virus has been dialyzed, use the same formulation as the final buffer.&nbsp; Use of MTN (0.6M NaCl, 0.1M Tris-Cl, pH 7.5, 0.1M MgCl2) that has been 0.02 &mu;m filtered works very well for many viruses (better than TE).<br /><br />Note: Avoid vortexing or pipetting virus samples up and down as this may cause capsids to burst and release DNA which will decrease virus count and cause messy background.

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TITLE: Measurement of cellular oxygen consumption and extracellular acidification
AUTHORS: Kiichi Hirota, Yoshiyuki Matsuo

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

[STEPS]
?. Cells were seeded on to the XFp Cell Culture microplate and incubated overnight.
?. The sensor cartridge of the XFp Analyzer was hydrated at 37 °C in a non-CO2 incubator one day before the experiment.
?. For the OCR assay, injection port A on the sensor cartridge was loaded with oligomycin (a complex V inhibitor, final concentration 1 µM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; an uncoupling agent, final concentration 2 µM) was loaded to port B, and rotenone/antimycin A (inhibitors of complexes I and III, final concentration 0.5 µM each) was loaded to port C.
?. During sensor calibration, cells were incubated at 37 °C in the non-CO2incubator in 180 μl assay medium (XF Base Medium: 25 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine, pH 7.4).
?. The plate was immediately placed on the calibrated XFp Analyzer for the assay.
TITLE: Free Floating IHC Fluorescent Staining 
AUTHORS: Ashley Harms, Jhodi Webster

[DESCRIPTION]
This protocol is optimized for to stain free floating sections for fluorescent labelling. Please note than each step must be done on a shaker. 

[STEPS]
SECTION: Day 1
1. Using a 12-well plate with buckets, wash sections 3x10min TBS
SECTION: Day 1
3. Block in 5% serum in TBST for 1hour at RT (using approx 2mL per well)
SECTION: Day 1
4. Using approx 500uL per well, let sections wash in primary antibody solution in TBST + 1% serum overnight in 4 °C 

SECTION: Day 2
5. Wash sections 3x10min in TBST

SECTION: Day 2
6. Incubate in secondary antibody (1:1000 for Invitrogen-brand or 1:2000 for BioRad-brand in TBST + 1%serum) for 2 hours @ RT in the dark. Approx 2mL per well. 
SECTION: Day 2
7. Wash 3x10min in TBS
SECTION: Day 2
8. Mount and allow slides to dry. Coverslip once sections are completely dried on the slide (. 
SECTION: Day 1
2. Depending on antibody of choice, do an antigen retrieval step. 
Antigen retrieval in TBS:     
10mM sodium citrate      
0.05% Tween-20
TITLE: Dark Light Box
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Behavioral test for anxiety-like behaviors in mice.

[STEPS]
1. Set up the dark light box. See materials. 
2. Place each mouse into the dark compartment before closing the lid and left to explore for 6
minutes. 
3. Video record each experiments.
4. Manually score for each animal the time in the light box as well as the number of times the mouse entered the light box.
TITLE: Preparing a Bacterial Stock 
AUTHORS: Wolfram Moebius, Dibyendu Dutta

[DESCRIPTION]
This protocol describes how to create a glycerol bacterial stock for long-term storage at -70 °C.



[STEPS]
SECTION: Preparing Bacterial Stock
1. Produce an   culture of the desired strain. 
SECTION: Preparing Bacterial Stock
2.    Label cryotube lid and side of tube with with strain, date. 

Create 30 % glycerol (3 parts glycerol by volume, 7 parts Millipore H2O by volume) then autoclave. 

SECTION: Preparing Bacterial Stock
4. Store at -80 °C . 

SECTION: Preparing Bacterial Stock
3. Make a 1:1 mix of 30 % glycerol and the O/N culture by adding 500 µL O/N culture to 500 µL 30% glycerol in the cryotube and gently mix via vortex.
TITLE: Spatial Transcriptomics for OCT using 10x Genomics Visium
AUTHORS: Andrew Houston, Siqi Chen, Feng Chen

[DESCRIPTION]
To detect gene expression spatially mapped across a fresh-frozen tissue sample adapted from 10x Genomics Visium protocol. 

[STEPS]
SECTION: Visium Fresh-Frozen
2. RNA extraction and assessment is outlined in the above guide but not always necessary
Most OCT blocks have preserved RNA quality well due to their preservation/storage
SECTION: Visium Fresh-Frozen
3. Perform all fixation/H&E staining/imaging steps on the Visium Spatial Gene Expression Slide (PN-2000233)
Utilize the guide here
Typically it is not necessary to utilize a coverslip while imaging

SECTION: Visium Fresh-Frozen
1. Start here
If performing OCT tissue embedding for the first time be sure to consult pgs.5-9
If utilizing a new tissue type for the first time be sure to utilize the Tissue Optimization Guide which can be found here
3. Immediately proceed to the Visium Library Construction
Start on pg. 35 using this guide here
4. 
SECTION: Visium Fresh-Frozen
4. Immediately proceed to the Visium Library Construction
Start on pg. 35 using this guide here
SECTION: Visium Fresh-Frozen
5. Sequence the libraries according to the requirements on pg. 59 of CG000239
TITLE: Yeast growth profile analysis
AUTHORS: Monica Rieth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This method outlines a procedure for analyzing the growth curve of Baker's yeast, </span><span style = "font-style:italic;">Saccharomyces cerevisiae</span><span>, for the assessment of growth medias, the addition of metabolites to media, and the incorporation of heterologous genes to our yeast strain.</span></div></div>

[STEPS]
TITLE: Ectoplasmic Net (EN) formation in Aurantiochytrium limacinum (ATCC MYA-1381)
AUTHORS: Anbarasu Karthikaichamy, Jackie Collier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for ectoplasmic net (EN) formation in </span><span style = "font-style:italic;">Aurantiochytrium limacinum</span><span> (ATCC MYA-1381). </span></div></div>

[STEPS]
?. [d-GPY plating]
Take  of 3 day old cells grown on GPY media and plate it on d-GPY plates.  AB1ComponentsQuantity (%)2Yeast extract0.053Peptone0.14Glucose0.25Prepare 1/2 saline media by adding appropriate amount of instant ocean and water.6For agar plates1.5% agarIncubate the plates at
150 µl
AB1ComponentsQuantity (%)2Yeast extract0.053Peptone0.14Glucose0.25Prepare 1/2 saline media by adding appropriate amount of instant ocean and water.6For agar plates1.5% agar
25 °C
Add appropriate antibiotics to minimize bacterial contamination.
?. [Zoospore production]
After overnight incubation (~24 h), add just enough artificial sea water ((with appropriate antibiotics) to cover the entire plate.Incubate the plates for      After the incubation, take a small aliquot from the sea water layer and observe for the presence of zoospore. The zoospore production starts in about 90 mins, but it peaks around 3 h.Incubate the plates for After the incubation, take a small aliquot from the sea water layer and observe for the presence of zoospore. The zoospore production starts in about 90 mins, but it peaks around 3 h.
25 °C
Do not disturb the plates!
?. [Settlement and EN formation]
Take an empty sterile petridish and place a sterile microscopic slide.Add just enough A1 media (with appropriate antibiotics) on to the petridish so that the microscopic slide is immersed. Alternatively, a cover slip can also be used.Aspirate arround    to   of zoospores without disturbing the plate.Carefully drop the zoospores all over the microscopic slide.Cover the petridish and incubate at       Observe the EN the next day.Aspirate arround   to  of zoospores without disturbing the plate.Carefully drop the zoospores all over the microscopic slide.Cover the petridish and incubate at       Observe the EN the next day.Cover the petridish and incubate at
50 µl
100 µl
25 °C
EN starts forming after 90 mins incubation in the slides. If the experiment demands to observe EN formation, the zoospores can be allowed to settle on a glass bottom petridish for overnight timelapse microscopy.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>FIGURE OUT WHAT YOU NEED:</strong><br /><br /> A. Type of gel: regular agarose or low melting point (LMP) agarose<br /><br /> B. Concentration of gel<br />       1. Mini gels (50 mL gels):<br />              0.7% = 0.35 gm per 50 mL<br />              0.8% = 0.40 gm per 50 mL<br />              1.2% = 0.60 gm per 50 mL<br />       2. Small gels (100 mL gels):<br />              0.7% = 0.70 gm per 100 mL<br />              0.8% = 0.80 gm per 100 mL<br />              1.2% = 1.20 gm per 100 mL<br />       3. Large gels (250 mL gels):<br />              0.7% = 1.75 gm per 250 mL<br />              0.8% = 2.00 gm per 250 mL<br />              1.2% = 3.00 gm per 250 mL<br /><br /> The concentration of the gel will depend on the size fragments you are working with. Low concentration gels are used for large DNA fragment and high concentration gels are used for small DNA fragments.<br /><br /> C. Type of buffer<br />       1. TPE (Tris/Phosphate/EDTA), 10X stock<br />       2. TBE (Tris/Borate/EDTA), 10X stock<br />       3. TAE (Tris/Acetate/EDTA), 10X stock<br /><br /> TPE buffer is a general use buffer. TBE and TAE buffers are generally used when electroeluting fragments from gels.<br /><br /> D. Size of gel<br />       1. Mini - 50 ml gel + 250 ml running buffer<br />       2. Small - 100 ml gel + 900 ml running buffer<br />       3. Large - 250 ml gel + 2000 ml running buffer (slope end gel boxes) or 2500 ml running buffer (square end gel boxes)<br /><br /> E. Gel combs<br />       1. Mini - 8 lane combs (to 25 μl each well)<br />       2. Small - 14 lane or 20 lane combs (to 25 μl each well)<br />       3. Large - 20 lane or 30 lane combs (to 20 μl each well)<br /><br /><strong>GEL BUFFERS AND ELECTROPHORESIS MATERIALS: </strong><br /><br />1) TPE buffer, 2 liters, 10X <br />       216.0 gm of Tris base <br />       30.2 mL of 85% Phosphoric acid <br />       80.0 mL of 500 mM EDTA, pH 8.0 <br /><br />2) TBE buffer, 2 liters, 10X <br />       216.0 gm of Tris base <br />       110.0 gm of Boric acid <br />       100.0 mL of 500 mM EDTA, pH 8.0 <br /><br />    Filter the 10X buffer through Whatman #1 filter paper before placing the buffer in the bottle. <br /><br />3) TAE buffer, 2 liters, 10X <br />       193.76 gm of Tris base <br />       108.86 gm of Sodium acetate <br />       26.9 gm of disodium EDTA <br /><br />    Adjust the pH to 8.0 with glacial acetic acid.

[STEPS]
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TITLE: Extraction of fungal DNA and PCR for identification using the Sigma-Aldrich REDExtract Plant PCR kit
AUTHORS: Siouxsie Wiles, Shara Van De Pas

[DESCRIPTION]
This protocol describes how we use the REDExtract Plant PCR kit (Sigma-Aldrich) to extract DNA from fungal mycelium and PCR from the ITS region.

[BEFORE_START]
You will need to prepare your fungal cultures so that you have visible mycelium for extraction.

[GUIDELINES]
Make sure your fungal plates aren't contaminated! 

[STEPS]
SECTION: DNA Extraction
1. Place 30 ul extraction solution into PCR tubes. Using a small sterile pipette tip place a small piece of mycelium so its only just visible on the tip into the tube. Vortex and briefly centrifuge.
SECTION: DNA Extraction
2. Place PCR tubes into the PCR machine and run a cell break protocol (95 °C for 10 minutes).
Cool down and add 30µl dilution solution. 
Dilute the samples as needed. Start with 1:20 and go to 1:10 or 1:5 if needed. 
Store the extracted DNA at  -20 °C until PCR.

SECTION: PCR
3. Make a master mix based on 9 µl per single reaction

Recipe per PCR sample (single reaction = 9 µl)

 

SECTION: PCR
5. Add 1µl of extracted DNA sample to each aliquot of master mix. 
SECTION: PCR
7. Place tubes into PCR machine and run using the following settings:

1x 94 °C - 3 minutes.

35-40x cycles of:
    94 °C - 30 seconds
    52 °C - 30 seconds
    72 °C - 30 seconds (For LSU primers use  72 °C  for 40 sec)  
 
1x 72 °C  - 7 minutes

SECTION: Gel electrophoresis
8. Make a 1% molecular grade agarose gel using 1x fresh TAE. Microwave until the agar has just dissolved.
Note. 100ml for running 25 samples, 35ml for 13 samples
SECTION: Gel electrophoresis
9. Add 2µL Sybrsafe stain per 50ml volume of agar
SECTION: Gel electrophoresis
10. Pour into setup gel tray, add comb and leave for half an hour or until set.
SECTION: Gel electrophoresis
11. Place the set gel into the gel tank (make sure the wells are at the negative end).
SECTION: Gel electrophoresis
12. Fill the tank with waste TAE until entire gel is submerged - can top up with fresh TAE if needed.
SECTION: Gel electrophoresis
13. Load 2-5µl of sample into each well including a 1kb ladder and negative controls
SECTION: Gel electrophoresis
14. Set powerpack to run at 90v for 30mins
SECTION: Gel electrophoresis
15. After gel is run gently lift the tray out of the tank and movie it onto a sybersafe tray view bands in the gel viewer.
SECTION: PCR
4. Aliquot 9µl of master mix into individual PCR tubes.
SECTION: PCR
6. Centrifuge samples briefly (just a few seconds) in a benchtop centrifuge to ensure they are at the bottom of the tube.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>sgRNA template assembly, in vitro T7 transcription, and SPRI bead cleanup</p>

[GUIDELINES]
The primers used are: one long, variable oligo that carries the T7 promoter and desired guide sequence; an 82-nt constant oligo that carries the 3' end of the sgRNA; two short external primers for amplification.<br /><strong><br />Assembly Oligos:</strong><br />T7FwdVar: <br />GGATCCTAATACGACTCACTATAG---guide-sequence---GTTTTAGAGCTAGAA <br />T7RevLong: AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC <br /><br /><strong>Amplification Primers:</strong><br />T7FwdAmp: GGATCCTAATACGACTCACTATAG <br />T7RevAmp: AAAAAAGCACCGACTCGG <br /> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/6zc75n.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/6yc75n.png" data-ofn="T7E1 gel.png" /><br /><strong>Designing the guide sequence:</strong><span style="font-weight: 400;">The T7 transcript starts with the G before the dashes in the Fwd assembly oligo. If your target calls for a G at the 5’ end of the guide, use the one already included in T7FwdVar design. If your target calls for a different base at the 5’ end, put the whole guide sequence in T7FwdVar; there will be an extra G on the end that should have minimal effect, unless your guide is quite short.<br /></span><strong>Making the template: </strong>we have used <span style="font-weight: 400;">the Phusion-HF DNA polymerase. Although the amplification primers have quite different Tm’s, they work together fine. </span><br /><br /><br />

[STEPS]
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