eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol for Using the Microfluidizer Lysis Apparatus
AUTHORS: Clark Fritsch

[DESCRIPTION]
The Microfluidizer Lysis apparatus is used to efficiently lyse bacterial and yeast cells using high pressure sheering against a ceramic cone inside the machine. Unlike conventional approaches for cell lysis such as sonication or French pressing, the microfluidizer is able to efficiently lyse your cells quickly and efficiently, only taking approximately 1 – 2 minutes. Cells can be passed through the Microfluidizer multiple times and the pressure of the machine can be adjusted based on cell type. Note, however, that the Microfluidizer does not work efficiently on mammalian cells.

[STEPS]
1. Begin by filling the red tray next to the Microfluidizer with a ice. Pack the ice densely around the sheering vessel/compartment, as shown in Figure 1. The sheering vessel flows your cells at ultrasonic speeds against a pointed ceramic cone. This quickly and efficiently sheers your cells, but it also heats your sample up a bit. To minimize 
heat exposure, you should make sure that as much of the vessel is packed tightly with ice prior to use. Further, after packing the shearing vessel with ice, let it cool for 20 – 30 minutes prior to use.

  
2. When you first use the Microfluidizer, you will see some liquid in the sample chamber, as shown in Figure 2. This is the storage solution – 70% isopropanol. Before you add your sample to the sample chamber, you will want to wash out the sample chamber extensively with water. To do so, flip the valve as shown in Figure 3. This will start the machine. Note that when you are washing the sample chamber, never let air get into the machine. You should never let the machine run if the chamber is empty/dry. Always make sure that liquid is flowing through the machine to prevent air bubbles from getting into the system.

  
  

3. After you have washed the sample chamber with water, you will want to equilibrate the valves with your sample buffer. You will want to collect the waste from your washes in a container below the sample chamber, as shown in Figure 2. When you are collecting your lysed sample, it is a good idea to have a container that is also chilled on ice.
4. After washing the sample chamber and equilibriated it with your Sample buffer, switch the waste vessel and place the chilled collection Beaker (on ice) under the collection nozzle.
5. Then begin to flow through your sample by slowly adding it to the sample chamber. Remember to never let the sample chamber become empty. After you have finished adding your sample to the sample chamber, add water to the sample chamber until the flow through is clear.
6. After you have collected your sheared sample, you may flow it through the Microfluidizer again by simply adding it back to the Sample Chamber. Once you have sheared your sample sufficiently, make sure to centrifuge it quickly to prevent degradation.
7. Clean the Microfluidizer by washing extensively with water. Then wash with 70% isopropanol and then store in 70% isopropanol and turn off the valve. This means that the Sample Chamber will be filled up with 70% isopropanol.
TITLE: U Cinn - Triglyceride Assay
AUTHORS: Patrick Tso, Dana Lee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">Determinations of triglycerides in plasma/serum/lymph will be made using a Randox Triglycerides colorimetric kit.  The triglycerides are determined after enzymatic hydrolysis with lipases.</div></div>

[STEPS]
?. Prepare Working Standards by making a serial dilution of the stock 200mg/dl standard. (See diagram below) *Stock standard is included in kit
?. Prepare Working Reagent by reconstituting one vial of Enzyme Reagent R1b with a portion of Buffer R1a and then transfer entire contents to bottle R1a, rinsing vial R1b several times.
?. Using a 96 well flat bottom plate, into separate wells, pipette 2μL of deionized water, standard, or sample to be assayed.
?. Add 200μL of reconstituted Working Reagent to all wells.
?. Incubate plate for 5 minutes at 37°C
?. Determine the absorbance (abs) of the standards and of each unknown at 500nm.
?. Calculate values of unknowns from the standard curve.Specimen:  Serum or Plasma.  Specimen stable for 7 days at 2-8°C or 3 months at -20°C. Assay Linearity: 1172 mg/dl Working Reagent Stability After Reconstitution: 21 days stored at 2-8°C. PROTECT FROM LIGHT Stability of Final Reaction:  60 minutes
TITLE: Modular automated sample processing of biological samples using ultrasonication and a workstation for high-throughput proteomics
AUTHORS: ronan.ocualain

[DESCRIPTION]
Sample preparation for mass spectrometry analysis involves numerous liquid transfer steps.

These include 
sample lysis, 
protein extraction, 
solubilisation, 
estimation, 
reduction and alkylation, 
normalisation, 
clean-up, 
enzymatic digestion, 
and desalting. 

Adapting these steps onto an automated workstation can increase efficiency, throughput, and reduce coefficients of variance (%CV) thereby providing reliable reproducible data for statistical comparisons.  

This protocol is part of a modular collection for the processing of biological samples for proteomics. 

The entry point is ultrasonication of biological samples (cells, tissues, laser captured FFPE sections) using a plate based LE220+ system from Covaris, followed by sample processing with a Biomek NxP workstation.

Technical measurement of workflow percentage coefficients of variation (%CVs) using HeLa extract at the entry stage, to measurement of the ion intensities at the data processing stage with data collected by DDA using a QE-HF and processed using Progenesis-QI for proteomics has shown that one third of peptides have %CVs below 20%, and with 80% of peptides having %CVs below 30%. Using Progenesis QI for proteomics indicates that 98% of peptides are found in all 7 replicates processed.


 


In addition, the whole procedure (80 samples) may be completed in a day, if shorter digestion times are utilised.

[STEPS]
TITLE:  Preparation of biological monolayers for producing high-resolution scanning electron micrographs Code: PLOS2021 [PONE-D-21-26669] - [EMID:f8569bc7b537f6e4]
AUTHORS: Shireen     Mentor, Franscious  Cummings, David  Fisher, shireen.mentor  

[DESCRIPTION]
Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, viz., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to grow an overnight culture of bacteria in LB (Luria-Bertani) liquid media broth. 

The overnight culture can be used directly, reinoculated into a larger volume of LB broth or plated to acquire either a lawn of bacterial growth or single isolated colonies.



[BEFORE_START]
Before starting this protocol users will need to have prepared LB broth. If instruction is required for making LB broth  please see the protocol 'LB (Luria-Bertani) liquid medium' by the same author.

How much LB broth you inoculate will depend on what you plan to do with the bacteria culture the next day? 
If only requiring a small volume of bacteria and a low optical density (i.e. OD <5) then only a small volume of LB broth (i.e. 10 mL) will be required as you will either dilute down the bacterial suspension or spin down and pellet the bacteria before re-suspension to achieve the desired optical density. 
However, if requiring a large volume of bacteria and/or a high optical density (i.e. OD <25), the following day you will need to re-inoculate the culture into a larger volume of LB broth, allow this to reach exponential growth phase and then spin down and pellet the bacteria, before re-suspension to reach the required O.D. If planning on re-inoculating the culture then the overnight culture volume should be 10% of the total volume you plan to re-inoculate (i.e. inoculate 100 mL LB broth if planning to re-inoculate 1 L the next day). 


[GUIDELINES]
Growing an overnight bacteria culture can be done by either-
Inoculating media with a single isolated colony of bacteria collected from a plate 
Inoculating media by adding a frozen glycerol-bacteria suspension aliquot grown from a single isolated colony 



[STEPS]
?. 
?. 
?. 
?.
TITLE: Bogus data acquisition protocol II
AUTHORS: Abby   Moore

[DESCRIPTION]
The purpose of this protocol is to demo protocol development.

[BEFORE_START]
These are my warnings.

[GUIDELINES]
These are my guidelines.        

[STEPS]
2. In this step, I'll use embed code for Box to display an image of software.

 

3. In this step, I'll use the command component to refer to a command.


 

1. Before starting, you need to run a test using the following protocol. I referred to this protocol using a hyperlink: Bogus Data Acquisition Protocol I.
TITLE: DAB precipitation by Laccase Oxidation 
AUTHORS: Song-Yi Lee, Heegwang Roh, Mason Mackey, Alice Y. Ting, Mark Ellisman

[DESCRIPTION]
Genetically-encoded oxidase, LaccID, oxidizes and precipitates DAB with oxygen and therefore without the need for H2O2 or light.  LaccID-expressing HEX293T cells precipitated DAB and were processed for TEM. 

[STEPS]
SECTION: Cell Fixation
1. LaccID-expressing HEX293T cells are plated onto MatTek dishes containing 35mm glass bottom No. 0 coverslips coated with poly-d-lysine (P35GC-0-14C, MatTek Corporation).
SECTION: Cell Fixation
3. Cells are washed five times for 2 min each on ice with cold 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4
SECTION: Cell Fixation
4. Cell are blocked for 5 min with cold 20 mM glycine in 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4
SECTION: Cell Fixation
5. A freshly solution of 5.4 mg of 3,3’- Diaminobenzidine (DAB) (D8001-10G, Sigma-Aldrich) is dissolved in 1.0 ml of 0.1 N HCl and added to 9.0 ml 0.1M sodium cacodylate buffer with 2 mM CaCl2, pH 7.4 buffer.     
SECTION: Cell Fixation
7. Cells are then washed five times for 2 min each with cold 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4.
SECTION: TEM processing
8. Cells are post-fixed with 1% osmium tetroxide (EMS, 19150) in 0.1 M sodium cacodylate buffer pH 7.4 for 30 min on ice. 
SECTION: TEM processing
9. Postfixative is removed and cells are washed (five times for 1 min each) with 0.1 M sodium cacodylate buffer pH 7.4 containing 2 mM CaCl2 on ice
SECTION: TEM processing
10. Cells are washed (five times for 1 min each) with cold double-distilled H2O (ddH2O) on ice.
SECTION: TEM processing
11. Cells are dehydrated with an ethanol series (20%, 50%, 70%, 90% and 100%) on ice for 1 min each.
SECTION: TEM processing
12. Cells are dehydrated with 100% dry ethanol at room temperature three times for 1 min each.
SECTION: TEM processing
13. Cells are infiltrated with 50:50 dry ethanol:Durcupan ACM resin (Sigma-Aldrich, 44610) for 30 min and then four changes of Durcupan at 1 h each and finally embedded in a vacuum oven at 60 °C for 72 hours.
SECTION: Cell Fixation
2. Cells are fixed using warm 2% glutaraldehyde (18426, Ted Pella Incorporated) in buffer 0.1M sodium cacodylate with 2 mM CaCl2, pH 7.4 (12310, Electron Microscopy Sciences) and then quickly moved to ice for 55 minutes.  
SECTION: Cell Fixation
6. The DAB solution is added to cells for at least 3-30 min at room temperature until the desired brown intensity color of the precipitate was visible.
TITLE: Traditional Chinese Medicine for Helicobacter Pylori infection: An Evidence Assessment for Systemic Reviews and Meta-analysesl
AUTHORS: Xiao-bei Si, Shuai Wang, Xu-min Zhang, Yu Lan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Background:</span><span> In recent years, many studies reported the anti-Hp effects of Traditional Chinese Medicine. Based on these studies, there had been many relevant systemic reviews and meta analyses with variable results</span></div><div class = "text-block"><span style = "font-style:italic;">Aim: </span><span>This overview aims to investigate the general characteristics and assess the methodological quality of these systemic reviews/meta ana lyses (SR/MAs).</span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Method:</span><span> In this overview, we intend to includ systemic reviews/meta analyses with comparison of Chinese medicine and western medicine on helicobacter pylori infection by searching databases of Medline, Embase, Web of Science, China National Knowledge Infrastructure Database (CNKI), Chinese VIP Information (VIP), Chinese Medical Databases (CMB) and Wan-Fang Database until December 16th, 2018. AMSTAR and PRISMA tools will be adopted to assess the methodological quality and reporting characteristics respectively. </span></div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">Conclusion:</span><span> The methodological quality of these SR/MAs will be assessed as clinical evidence and our result may help further use of them. </span></div></div>

[STEPS]
TITLE: Preparation of α-synuclein fibrils amplified from clinical material
AUTHORS: arpine.sokratian

[DESCRIPTION]
This protocol describes the preparation of alpha-synuclein fibrils from clinical tissue with a quality control. 
Optional conjugation step with phRodo STP ester dye is indicated

[STEPS]
1. Thaw down an aliquot of alpha-synuclein monomer stock (track down a batch number with identified EU number, concentration, A260/280 ratio) on ice
2. Spin down an aliquot of alpha-synuclein monomer stock solution ( , 10 min4 °C ) and measure the concentration using nanodrop 

Add 3 µL of 10x diluted aliquot in PBS onto nanodrop pedestal; 
Parameters: other proteins; coefficient extinction: 5.98; MW: 14.4 kDA
Perform two measurements and confirm <10% standard error between two measurements
If necessary, prepare 20X and 30X dilutions to confirm findings. 
 


3. Calculate the reaction mix: 5 mg/mL concentration of alpha-synuclein in 200 µL in PBS (use safe-lock eppendorf tubes or other  )

4. Incubate the reaction at  for 7200 min using program settings: 1 min ON; 1 min OFF
 

 



5. Spin down the insoluble fraction at 15000 rpm, 10 min, 10 °C; take 100 µL of supernatant and add 1 mL of fresh PBS to the pellet

 

6. Gently resuspend and spin down the fibrils at 15000 rpm, 10 min, 10 °C; take 100 µL of supernatant and add 1 mL of fresh PBS to the pellet (repeat 3 times)
7. 
9. Sonicate the fibrils using water bath sonicator at 10C, 30% amplitude and for 1 hour (no OFF ON cycles). Check the level of water in the water-bath is in a line with the tube content. 
10. Measure size of sonicated particles using DLS 
 

3.1. Prepare a reaction mix containing 10% of clinical tissue and 5 mg/mL of alpha-synuclein in PBS

8. Take out 900 ul of PBS leaving 100 ul of fibril pellet in the tubes. Transfer the fibrils into 0.6 mL PCR tubes (thick wall).
TITLE: SPARC Cat - Sham Control Chronic Cat 3 Day 30
AUTHORS: Brett             Hanzlicek, Anna Rietsch, Margot Damaser

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a procedure for a sham control chronic cat experiment (Day 30-terminal) for cystotomy (bladder surgery).  The cystotomy is performed without UroMOCA implantation.  The cat is observed daily and imaged on day 0, 14 and 30 to track changes to the bladder and overall cat health in response to the cystotomy.  This protocol includes urodynamics and imaging for Day 30 in the chronic experiments.</div></div>

[STEPS]
?. [Transport Cat]
Transport cat from housing site to surgery site.
?. [Animal Prep and catheter placement]
Animal is anesthetized and abdomen is shaved by the vet team.  The cat is then moved into the surgery room and attached to monitors by the vet team.
?. [Animal Prep and catheter placement]
Drape animal and perform betadine scrub on abdomen and genitals.
?. [Animal Prep and catheter placement]
Put surgilube on 3.5Fr catheter and insert into bladder through the urethra.  Advance the catheter until resistance is met, then pull back 2-3cm.
?. [Animal Prep and catheter placement]
Use gentle suction with syringe to withdraw urine from bladder through the catheter.  Measure the volume and save the urine for urinalysis.  of urine was removed
18 mL
?. [Cystogram - DYNA CT]
Use 1:5 dilution of contrast to saline to visualize bladder.Fill the bladder with   contrastand take a single imageAdd   more (for a total of 20ml) and take a single imageAdd   more (for a total of 25ml) and take a single imageTake a 3D CT image with 25ml contrast:saline in the bladder
10 mL
10 mL
5 mL
?. [Cystogram - DYNA CT]
Empty Bladder
?. [Urodynamics/Cystometry 1]
Performed after cat is transferred from Isoflurane to propofol anesthetic.
Fill rate is 2ml saline/minute using syringe pump.
Data is recorded using Labview software.
Pressure is recorded using an external pressure transducer connected to syringe pump on one end and tubing that leads to the catheter on the other end.
?. [Urodynamics/Cystometry 1]
Fill bladder with saline and record pressure using pressure transducer connected to  LabViewFill to
20 mL
Pressure very high. Recalibrate and go to cystometry 2.
?. [Urodynamics/Cystometry 1]
Empty Bladder
?. [Urodynamics/cystometry 2]
Fill bladder with saline at 2ml/min
?. [Urodynamics/cystometry 2]
Fill to  ; End round 2 urodynamics
20 mL
Strange curve – no pressure change when tugging on the catheter.   Likely a clot in the bladder from possible scraping of the bladder.  End experiment.
?. [Dissection/Histology]
Bladder is removed, examined and placed in formalin for histological analysis
TITLE: Frailty in European Emergency Departments (the FEED study)
AUTHORS: James D van Oppen

[DESCRIPTION]
This study will describe the prevalence of frailty and methods in use for its assessment in European emergency departments. This knowledge will inform population-attuned configuration of healthcare services and education. 

[STEPS]
1. Survey to determine site characteristics and availability of frailty-attuned healthcare services.
2. Cross-sectional study to determine the prevalence of older age and frailty in emergency care.
TITLE: Protocol for &#34;Neuromelanin accumulation drives endogenous synuclienopathy in non-human primates&#34;
AUTHORS: jlanciego

[DESCRIPTION]
This study was aimed to develop and characterize
a non-human primate (NHP) model of Parkinson’s disease mimicking the known
neuropathological hallmarks of Parkinson’s disease to the best possible extent.
Accordingly, we sought to determine whether AAV-mediated enhanced expression of
human tyrosinase (hTyr) in the substantia nigra (SNpc) of non-human primates
(NHPs) is able to induce a time-dependent accumulation of neuromelanin (NMel)
in dopaminergic neurons, further triggering and endogenous synucleinopathy,
progressive cell death and a pro-inflammatory scenario, in keeping with what
was formerly reported in rats by taking advantage of a similar strategy (Carballo-Carbajal
et al., 2019). Furthermore, the potential prionoid spread of endogenous
alpha-synuclein (a-Syn) species towards the
prefrontal cortex was analyzed, in an attempt to evaluate to what extent there
is a propagation of endogenous a-Syn
by permissive trans-synaptic templating (e.g. the so-called Braak hypothesis).
Adult juvenile NHPs (Macaca fascicularis)
were injected with adeno-associated viral vectors (AAVs) encoding either the
hTyr gene (AAV-hTyr; delivered into the left SNpc) or a null construct for
control purposes (AAV-null; injected into the right SNpc). In order to
delineate a timeline for the underlying processes, one group of NHPs was
sacrificed four months post-AAV deliveries (animals M308F4 and M310M4), whereby
the follow-up timing for second experimental group was settled at eight months
post-AAVs surgeries (animals M307F8 and M309M8). Neuroimage studies (MRI and
MicroPET) were conducted in vivo at
different time points. Upon animal sacrifices, brain tissue samples were
processed for histological analysis comprising intracellular NMel levels,
intracellular aggregates, nigrostriatal degeneration and neuroinflammation.


[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A step-by-step procedure for autologous platelet-rich plasma production was developed for topical percutaneous injection in donkeys.</p>
<p>This protocol was used in the following publication:</p>
<p>Faillace V, Tambella AM, Fratini M, Paggi E, Dini F, Laus F. Use of autologous platelet-rich plasma for a delayed consolidation of a tibial fracture in a young donkey. <em>The Journal of Veterinary Medical Science</em>, 79(3), 2017: 618-622. (ISSN: 0916-7250) (DOI: 10.1292/jvms.16-0400)</p>
<p><a href="https://doi.org/10.1292/jvms.16-0400" target="_blank">https://doi.org/10.1292/jvms.16-0400</a></p>
<p><a href="https://www.jstage.jst.go.jp/article/jvms/79/3/79_16-0400/_pdf/-char/en" target="_blank">https://www.jstage.jst.go.jp/article/jvms/79/3/79_16-0400/_pdf/-char/en</a></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.