eamag/protocols-with-steps huggingface dataset:
TITLE: Ancient dental calculus sampling procedure for optical microscopic analysis
AUTHORS: Dulce Neves, Emanuela Cristiani, António Faustino Carvalho, Ana Maria Silva

[DESCRIPTION]
This protocol describes how to sample dental calculus from individual teeth for optical microscopy analysis. The primary use-case is for the analysis of dental micro-remains embedded in the matrix of the dental calculus (e.g. starches, phytoliths, pollens, fungi, fibers, etc.), though it can also be used for biomolecular analysis (DNA and proteomics).

[GUIDELINES]
A clean sterile sampling environment is a vital factor to reduce contamination.

Gloves should be worn during the whole sampling procedure.

[STEPS]
SECTION: Sampling preparation and dental calculus collection
8. Display aluminum foil on the working table and carefully collect the dental calculus deposit using a sterile disposable blade and under the magnifying lens

 

SECTION: Sampling preparation and dental calculus collection
1. Clean the working table surface using alcohol and paper towels, and use powder-free nitrile gloves throughout the whole steps
SECTION: Sampling preparation and dental calculus collection
2. Make sure to sterilize all utensils used before starting the sample collection and between sampling each tooth. Use alcohol and the paper towels
SECTION: Sampling preparation and dental calculus collection
3. Cover the surface with plastic wrap
SECTION: Sampling preparation and dental calculus collection
4. Label the centrifuge tube ready for sampling
SECTION: Sampling preparation and dental calculus collection
5. Fill in the metadata sheet with all the information and the corresponding tubes IDs. Record the dental pathologies (Hillson, 2001), dental wear (Smith, 1984) and describe the deposit of dental calculus accordingly to its type and size (Buikstra and Ubelaker, 1994)

 

 

 

 


SECTION: Sampling preparation and dental calculus collection
6. Measure the dental calculus deposits using the Vernier caliper
SECTION: Sampling preparation and dental calculus collection
7. Photograph the tooth surfaces before collecting the dental calculus deposit
SECTION: Sampling preparation and dental calculus collection
10. Remove and discard the piece of foil
SECTION: Sampling preparation and dental calculus collection
9. Put the sample in the labelled centrifuge tube
SECTION: Sampling preparation and dental calculus collection
11. Weight the deposit using a precision scale
SECTION: Sampling preparation and dental calculus collection
12. Take new photos of the tooth after the collection of the dental calculus deposit
SECTION: Control samples
13. Take control samples of the working table surface (and other relevant surfaces) and maxillary bone (if present) to cross-out possible contamination
TITLE: MGH Harvard SenNet Processing murine trachea for paired single cell RNA-seq and mass spec
AUTHORS: gshipkovenska

[DESCRIPTION]
Protocol for obtaining single cell suspension of murine trachea.

[STEPS]
SECTION: Dissection
2. Perfuse lungs via right ventricle.
SECTION: Dissection
3. Dissect out trachea and lungs and place in HBSS on ice.
SECTION: Dissection
1. Euthanize mice by CO2 method.
SECTION: Dissection
4. Clean up the tracheas under the dissection microscope.
SECTION: Dissection
5. Bi-sect the tracheas along the ventral side.
SECTION: Single cell suspension
6.  Incubate each trachea in 500ul enzyme dissociation mix #1 for 30min with rotation  at 37*C.

Enzyme dissociation mix #1
1ml of papain
1ml of activation solution
20ul Dnase I 
SECTION: Single cell suspension
7. Cut off the tip of p1000 tip and pipet trachea up and down 10 times. Should get stringy.
SECTION: Single cell suspension
8. Spin down for 5 min at 2000g. This will pellet both single cells and the husk.
SECTION: Single cell suspension
9. Using a pipet (not the vacuum), remove as much supernatant as possible without being a hero.
SECTION: Single cell suspension
10. Resuspend cells and husk in 500ul enzyme mix #2 per trachea for 20 minutes at 37C with rocking.

Enzyme dissociation mix #2
25ul of 70kU/ml Collagenase I (in DMEM/ringers) 
25ul of 50kU/ml hyluronidase (in DMEM/ringers)
50ul of 7.5kU/ml DNAse (in DMEM/ringers)
120ul of 2.5U/ml dispase (in DMEM/ringers)
400ul of 2X papain (40 u/ml) (in EBSS)
QC to 5 ml of DMEM (has Ca and Mg, so good to use).
SECTION: Single cell suspension
11. Cut off the tip of p1000 tip and pipet trachea up and down 10 times. Husk will be mostly gone.
SECTION: Single cell suspension
12. Spike in 55ul FBS for 10% FBS final concentration to inactivate papain and mix well.
SECTION: Single cell suspension
13. Strain cells through 100um strainer.
SECTION: Single cell suspension
14. Centrifuge at 8000 × g for 2.5 min, discard the supernatant.
SECTION: Single cell suspension
15. Add 300 uL of RBC lysis buffer per trachea and incubate for 60s on the bench.
SECTION: Single cell suspension
16. Add 1 mL of EBSS to stop the lysing, centrifuge at 8000 × g for 2.5 min, discard the supernatant.
SECTION: Single cell suspension
17. Resuspend with 200 uL of 0.5% BSA in PBS. Keep on ice.
SECTION: Count cells with heamocytometer
18. Count two samples each per trachea. We routinely recover 250,000-350,000 cells with this protocol.



SECTION: Prepare samples for scRNA-seq and scMS
19. Split each tracheal sample into 50,000 cell aliquots, according to the cell counts determined above.
SECTION: Prepare samples for scRNA-seq and scMS
20. Keep one aliquot on ice for Andrew to pick up. This is the scRNA-seq sample.
SECTION: Prepare samples for scRNA-seq and scMS
21. For the remaining 3-4 aliquots, spin down at 8000 × g for 5 min, discard the supernatant and resuspend in 90% FBS, 10% DMSO and freeze at -80*C. These are the scMS samples.
SECTION: scRNA library preparation
22. Prepare according to manufacturer's protocol:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Detailed cell electroporation protocol from paper:</p>
<p><em>Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots</em><br />Eugene A Katrukha, Marina Mikhaylova, Hugo X van Brakel, Paul M van Bergen en Henegouwen, Anna Akhmanova, Casper C Hoogenraad, Lukas C Kapitein</p>

[GUIDELINES]
<p>From our experience, the number of incorporated QDs per cell mainly is proportional to the pouring pulse voltage, length (duration) and repetition. At the same time increase in these values leads to the overall decreased cell survival.</p>
<p>Another improvement can come from increasing QD concentration in the final electroporation solution. For example, by reducing Ringer's volume. But in this case air discharge can happen if electrodes are not immersed in solution.</p>
<p>18mm coverslips and 12 well cell culture plates can be also used.</p>
<p> </p>
<p>General advices if using different electroporator:</p>
<p>Specific CUY900-13-3-5 cell-culture-plate electrode has 5 mm distance between electrodes. What is important, is voltage per cm value. In our case, for pouring voltage V=200 V, l=0.5 cm, i.e. V/l = 400 V/cm.</p>

[STEPS]
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TITLE: Green Otter CBD Gummies Reviews - How It Works
AUTHORS: greenotterreviews  

[DESCRIPTION]
Green Otter CBD Gummies Reviews - How It Works


[STEPS]
1. Green Otter CBD Gummies Reviews - How It Works
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TITLE: Electromyogram recordings for internal capsule stimulations in non-human primates
AUTHORS: Lucy Liang, Elvira Pirondini, Jonathan C Ho

[DESCRIPTION]
EMG recordings is a common method to confirm implant location. In this protocol, we describe steps to perform EMG recordings during an internal capsule stimulator implant surgery. This procedure maybe performed in both sterile and non-sterile surgeries, but here we only describe the steps for a non-sterile, terminal experiment setup.

For step by step guidelines to plan an internal capsule implant surgery with the clinical robot, ROSA ONE Brain, refer to this protocol: dx.doi.org/10.17504/protocols.io.5jyl82rb7l2w/v1

This protocol is supplementary to the manuscript: 
Liang, L., Zimmermann Rollin, I., Alikaya, A., Ho, J.C., Santini, T., Bostan, A.C., Schwerdt, H.N., Stauffer, W.R., Ibrahim, T.S., Pirondini, E., Schaeffer, D.J., 2024. An open-source MRI compatible frame for multimodal presurgical mapping in macaque and capuchin monkeys. BioRxiv https://doi.org/10.1101/2024.02.17.580767

[BEFORE_START]
The animal should be under propofol/fentanyl i.v. (or any other anesthetics that allows for synaptic transmission) anesthesia before any step of this protocol is performed. Consult your veterinarian for details.

This protocols describes only the EMG procedure and assumes there is already an electrode placed within the hand area of the internal capsule of the animal. 

[STEPS]
SECTION: Needle electrode implants
1. Make a small incision on the forearm opposite to the side of the internal capsule (IC) electrode implant. 
SECTION: Needle electrode implants
2. Locate the flexor carpi radialis and/or any other muscle of interest, stimulate at around 1mA, 1Hz with bipolar probe electrode to confirm muscle.
SECTION: IC stimulation &amp; EMG recording
7. Stimulate the IC at 1Hz or 2Hz with continuous and biphasic pulses, and in the range of 0.8-4.8mA, depending on the motor threshold of the animal.
SECTION: Needle electrode implants
3. Place two 13mm needle electrodes inside, but along the belly of the muscle for differential EMG recording.
SECTION: Needle electrode implants
6. Repeat for upper arm, hand, and face muscles, or any other muscles of interest. Incision and stapling are not necessary for face muscles.
SECTION: Needle electrode implants
5. Close the incision with staples and tape the electrode cables to the arm. This will prevent them from being pulled out by accident.
SECTION: Needle electrode implants
4. Apply a few drops of Vetbond tissue glue to help the needles stay in place.
SECTION: IC stimulation &amp; EMG recording
8. If no movement was observed during stimulation, adjust the depth of the IC electrode slightly (+/- 2mm) to get a better activation.
SECTION: IC stimulation &amp; EMG recording
9. Once movement is observed, bring the stimulation amplitude down to motor threshold, record EMG with Ripple Neuro Trellis (or equivalent, at at least 2k samples per second).
SECTION: IC stimulation &amp; EMG recording
10. Check that the EMG activation is localized to the hand muscles.
TITLE: Chemical degradation and determination of pheomelanin and eumelanin markers
AUTHORS: Kazumasa Wakamatsu

[DESCRIPTION]
This is protocol involving chemical oxidation and reduction methods followed by high performance liquid chromatography (HPLC) detection of markers for pheomelanin and eumelanin.

[STEPS]
1. Homogenize samples in water with Ten-Broeck glass homogenizer at a concentration of 10 mg/mL (if samples were < 5 mg, use 0.5 mL of water).
2. 100 µL aliquots are then subjected to the chemical reactions.
3. Oxidation: Oxidize samples with 1.5% H2O2/K2CO3. After termination of reaction, leave mixtures for 20 hours at 25ºC (inducing secondary production of PTCA, PDCA, and TTCA)
4. Reduction: Heat samples with 57% HI in presence of H3PO2 at 130ºC for 20 hours. Analyze products, 4-AHP and 4-AHPEA
5. Levels of 4-AHP, the degradative product of DOPA pheomelanin, and 4-AHPEA, the degradative product of DA pheomelanin were analyzed by HPLC-ECD.
TITLE: A systematic review of the barriers to and facilitators of the use of evidence utilised by philanthropists when determining which charities, including health charities or programmes to fund.
AUTHORS: Caroline Greenhalgh, Paul Montgomery

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Today there is increasing recognition amongst philanthropists, charity leaders and policy makers, that in order to be effective, the process of giving resources needs to be grounded in evidence.  However, it is often suggested that there is insufficient evidence upon which philanthropists can rely, in part because ‘</span><span style = "font-style:italic;">good quality data on successful interventions is still not widely published’ </span><span>(Kassatly, 2018 p.1).  </span></div><div class = "text-block">This review aims to identify limiting and promoting factors regarding the use of evidence by philanthropists and to evaluate and synthesise existing research on this issue.  </div><div class = "text-block">This will include: </div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Primary research or systematic reviews investigating the perceptions and/or experiences of philanthropists relating to their use of evidence.  </li><li style = "counter-reset:ol0;list-style-type:disc;">Studies investigating the perceptions of professionals (such as charity CEO’s; philanthropic advisors and philanthropic consultants) as to how evidence is used by philanthropists. </li></ul></div><div class = "text-block">All study designs will be eligible for inclusion provided they examine factors affecting the use of evidence by philanthropists however, such factors need not be the primary focus of those studies.  </div><div class = "text-block">Anticipated findings relating to barriers include: insufficient time and resources to uncover relevant information; insufficient robust evidence; and a lack of trust in the sources of the information.  Expected facilitators of the use of evidence include: peer recommendations; conferences and philanthropy clubs or intermediaries.</div></div>

[STEPS]
TITLE: SDS-PAGE Using TGX Acrylamide Kit--CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prepare and run your SDS-PAGE gel using the Bio-Rad TGX gel system and the mini-PROTEAN gel box.</div></div>

[STEPS]
?. [Prepare the gel solutions and pour the gel]
Prepare the resolving gel acrylamide solution by combining 2 mL of Resolver A & B solutions in a 15 mL tube.
?. [Prepare the gel solutions and pour the gel]
Add 2 µL of TEMED and 20 µL of freshly prepared 10% w/v APS to the combined resolver solution and mix well. Use an appropriate pipette to steadily dispense the solution into the prepared gel cassette. Do not let bubbles form of solution mix with air. Fill the cassette to 0.5-1.0 cm below the bottom of the teeth on the comb. Immediately prepare and pour the stacking gel solutions as directed in the next steps.
You can quickly prepare a fresh solution of APS by adding approximately 100 mg of APS to a microcentrifuge tube, then adding approximately 1000 µL if ddH2O.
?. [Prepare the gel solutions and pour the gel]
Prepare the stacking gel acrylamide solution by combining 1 mL of the stacker A & B solutions in a 15 mL tube.
?. [Prepare the gel solutions and pour the gel]
Add 2 µL of TEMED and 10 µL of the freshly prepared 10% w/v APS to the combined stacker solution and mix well. Pipet solution down the middle of the gel cassette, filling to the top of the short plate. Apply slowly and steadily to prevent mixing with the resolving solution. Align and insert the comb in the cassette carefully to prevent air from being trapped under the comb teeth. Allow the gel to polymerize for 30-45 min before electrophoresis.
?. [Assemble the gel cassette]
Assemble the gel cassette on the casting stand. We are pouring a 0.75 mm thick gel and the volumes in this protocol are correct for this thickness of the gel.Watch this YouTube video to familiarize yourself with how to assemble and pour your gel. TGX Stain-Free™ FastCast™ Acrylamide Gel Casting - YouTube
?. [Prepare your samples and run your gel]
Use Laemmli sample buffer (4x) to prepare your samples. Your sample wells should hold up to 33 µL. You do not need to add sample buffer to the protein ladder, you just add the 5 µL aliquot of the protein ladder directly to a lane on your gel.
?. [Prepare your samples and run your gel]
Load your gel into the gel box. Here is a YouTube link to watch the process. Quick Tips: How to Load a Mini-PROTEAN TGX Stain-Free Gel for Electrophoresis - YouTube There are new and old gel boxes, so if you are using an older model, you can ask the TA or the Instructor for assistance.Fill the inner chamber completely with 1x Running Buffer and the outer chamber to the line for the number of gels you are planning to run.Run your gel between 100-200 V or until the loading dye has just run off the bottom of the gel.
TITLE: DNA Quantification -- CHEM 584
AUTHORS: Alba Balletbó, Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol adapted from the NanoDrop Spectrophotometer User’s Manual. </div></div>

[STEPS]
?. Start a new experiment using the Nanodrop touchscreen.
?. Click on the corresponding application module (e.g., dsDNA).
?. Open the sampling arm and load a blank sample (e.g., TE Buffer, MQ Water, etc.).
2 µl
?. Close the sampling arm on the machine to cover the blank sample.
?. Click "OK" to read the blank if the Nanodrop is not already in automatic mode.
?. Open the sampling arm and clean the blank off the upper and lower pedestals using a Kim Wipe.
?. Load your DNA sample and close the sampling arm.
2 µl
?. Click "Measure sample" if the Nanodrop is not in automatic mode.
?. Take a picture of the screen that shows your results and the corresponding spectra for your records.
?. Clean the nucleic acid sample off of the upper and lower pedestals using a Kim Wipe.
?. Repeat for all samples.
?. Close program.
TITLE: Binding Assay (His-tagged proteins)
AUTHORS: Terran D Stenger, Philippa R Kennedy

[DESCRIPTION]
To assess the binding specificity of tri-specific killer engagers (TriKEs) to their intended target, we utilize a flow cytometry-based binding assay leveraging the 10x polyhistidine (His) tag present on the TriKE construct, as previously described by our lab (see references). This assay enables assessment of TriKE binding efficiency and specificity, providing insights into TriKE-antigen interactions that inform further optimization and functional characterization of these therapeutic molecules.
 


[GUIDELINES]
Processed tissue additional controls: 
Fc Receptor Blocking Solution, cat. 422302, BioLegend, (1/100 dilution)

[STEPS]
SECTION: Live/Dead Staining and Fc Block
3. Prepare a solution of L/D NIR
SECTION: Cell Preparation
1. Cells are resuspended in R10 at 1.5 million cells/mL, and 200 µL (300k cells) are plated in each well in a U bottom 96 well plate.
SECTION: Cell Preparation
2. 300 x g, 5 min The plate is spun in a centrifuge at 300g for 5 min (this is approximately 1200rpm in a large benchtop centrifuge). The supernatant is removed.

SECTION: Preparation of Target Cells
2. 
SECTION: Incubation with His-Tagged Protein
3. 50 µL of 4X histidine(His)-tagged treatments are added to the pellet. 
SECTION: Incubation with His-Tagged Protein
4. 150 µL of R10 is added. Resuspend.
SECTION: Incubation with His-Tagged Protein
5. Incubate for 30 min at 37°C. 
SECTION: Extracellular + Live/Dead Staining
7. Cells are stained in 50 µL of flow buffer (1% AB serum, 0.5 mM EDTA in PBS) containing:
.05 µL LIVE/DEAD Fixable Near-IR Dead Cell Stain, cat. L34976, ThermoFisher
5 µL PE anti-His Tag Antibody, cat. 362603, Biolegend

Note: As controls, untreated cells are stained with the anti-His antibody, and TriKE-treated cells are stained with an isotype control antibody.
SECTION: Extracellular + Live/Dead Staining
8. Incubate for 20 min at 4°C. After the incubation, samples are washed twice with flow buffer to remove excess antibody.
SECTION: Extracellular + Live/Dead Staining
9. Cells are resuspended in 100 µL of 2% paraformaldehyde/PBS and incubated at room temperature in the dark for 10 min to fix them. Afterwards, wells are topped up to 200 µL with flow buffer and washed once. 
SECTION: Incubation with His-Tagged Protein
3.1. Recommended final concentrations: 0.3 nM, 3 nM, 30 nM for TriKEs and 3 nM, 30 nM and 300 nM for TriKE-PACC.
SECTION: Incubation with His-Tagged Protein
3.2. Prepare 4X drug in R10. 
SECTION: Incubation with His-Tagged Protein
6. After the incubation, samples are washed twice with flow buffer to remove excess, unbound protein.
SECTION: Flow Cytometry
10. Samples are analyzed via flow cytometry.
SECTION: Flow Cytometry
10.1.