eamag/protocols-with-steps huggingface dataset:
TITLE: How to request an assisted sorting session for the first time
AUTHORS: Daniel Gimenes

[DESCRIPTION]
Step by step protocol to create a PPMS account and to find the "Preliminary Sorting Discussion" form.

[STEPS]
SECTION: Creating a PPMS account
1. If you already have a PPMS account, please proceed to the step 6. If not, access the following link: https://ppms.embl.de/start/
SECTION: Creating a PPMS account
2. Click on Flow Cytometry Core Facility

 

SECTION: Creating a PPMS account
3. Click on "user account creation request".

 

SECTION: Creating a PPMS account
4. Next, you will be prompted to enter your credentials.
On the "Username" field, please fill with your username without the "@embl.de" (don't use your full email). After that, just use your regular login password

 

SECTION: Creating a PPMS account
5. You will be brought to a small form where you can fill the important information for the service billing and once submitted, the staff will receive an account creation request. Once we accept it, you should receive a confirmation by email. 🙂
SECTION: Requesting an assisted sorting
8. Login into your account

 

SECTION: Requesting an assisted sorting
13. To book a instrument, refer to the How to book a sorting slot in PPMS protocol
SECTION: Requesting an assisted sorting
12. Facility staff will receive your order, accept it and will check if everything is OK. If potential questions arises, the staff will send them to the user or will ask to schedule a meeting. 
When all the questions are clarified, the staff will give the users rights to book the instrument(s).
SECTION: Requesting an assisted sorting
11. Once you are done filling all the required information, click on "Submit Form".
SECTION: Requesting an assisted sorting
10. Optional: Attach the order to a project if available
Select a financial account for the order or keep the default one (Note that this service is not going to be charged!)
Add 1 to the "Quantity" box for the Preliminary Cell Sorting Request
Click on "Order".

 

SECTION: Requesting an assisted sorting
9. On the upper tabs, click on "ORDER"

 

SECTION: Requesting an assisted sorting
7. Click on Flow Cytometry Core Facility

 

SECTION: Requesting an assisted sorting
6. Go to https://ppms.embl.de
TITLE: Deposition of matrix using an M5 TM sprayer for high resolution MALDI analysis
AUTHORS: Martin Dufresne, Angela  Kruse, Jamie Allen, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
This protocol describes the use of an HTX M5 TM sprayer to deposit small molecule matrices onto tissue sections for high resolution MALDI imaging mass spectrometry (IMS). Spraying conditions will vary depending on matrix, and this protocol focuses on matrices for lipid IMS.

[STEPS]
SECTION: Autofluorescence Scan
1. Remove slides from freezer and place in desiccator for 30 min 

SECTION: Autofluorescence Scan
2. Collect autofluorescence scan using a Zeiss AxioScan Z1: Pre-IMS Autofluorescence Microscopy
SECTION: TM Sprayer Setup
3. Set nitrogen flow to 8 psi. turn on TM sprayer. 
SECTION: TM Sprayer Setup
4. Open HTX software
SECTION: TM Sprayer Setup
5. Set nozzle temperature to 60C, and heated stage temperature (if applicable) to 45C.
SECTION: TM Sprayer Setup
6. Clean sample loop with 10mL of methanol with sprayer set to "load". Switch to "spray" and allow to spray for 2 minutes.
SECTION: TM Sprayer Setup
7. Make matrix solution at a concentration of 12mg/mL in a solution of 60% THF and 40% acetone containing 5uL/mL DMF. Sonicate solution for 10 minutes.

Note that the matrix concentration and solvent composition may vary by sample and matrix. 
SECTION: TM Sprayer Setup
8. Switch sprayer to "load" and load sample loop with 2 mL of matrix solution using a syringe.
SECTION: TM Sprayer Setup
9. Switch sprayer to "spray" and allow to spray for ~30 seconds. Check that matrix is spraying by placing a glass slide under the nozzle. 
SECTION: TM Sprayer Setup
10. Tape slide onto heated stage, and adjust the spray area in the HTX software. 
SECTION: TM Sprayer Setup
11. Set the method in the HTX software:
1350 mm/min nozzle velocity
1.5 mm track spacing
0.05 mL/min flow rate
CC pattern
4 passes
40 mm nozzle height
No drying time
SECTION: TM Sprayer Setup
12. Ensure that the method is highlighted. Press start under the Cycle tab. Click Continue. Wait for purge delay and confirm that the nozzle is moving
SECTION: TM Sprayer Setup
13. When matrix deposition is finished, remove the slides and store them in a slide box. Press "valve load confirm" in the software.
SECTION: Cleanup
14. Set TM sprayer nozzle and stage temperatures to 30C
SECTION: Cleanup
15. With sprayer set to "load", load loop with 10 mL each of acetonitrile followed by methanol. Set sprayer to "spray" and allow to flow for ~5 minutes. 
SECTION: Cleanup
16. Spray nozzle tip with methanol, taking care not to touch the tip. 
SECTION: Cleanup
17. Clean the stage with methanol. Do not spray solvents directly onto the stage. 
SECTION: Cleanup
18. Replace the bench liner in front of the instruments
SECTION: Cleanup
19. Dispose of syringes in biohazard container
SECTION: Cleanup
20. Shut down HTX software and close the computer. Turn off the TM sprayer.
SECTION: Cleanup
21. Shut off the nitrogen flow.
TITLE: Multiplex Nested PCR for Vibrio Cholera
AUTHORS: Shannon Fitz, Alex Shaw, Dilip Abraham, michael Owusu

[DESCRIPTION]
Primers targeting various regions of the V. Cholera genome were obtained from the literature or designed, and optimized for a multiplex nested PCR assay. 

The following primers were extracted from Hoshino et al., 1998: O1_rfbFor_Inner (modified here by Dr Alex Shaw), O1_rfbRev_Inner, O139_rfbFor_Inner, O139_rfbRev_Outer. 

The following primers were obtained from Theron et al., 2000: ctxA Inner Rev, ctxA_rev. 

The following primers were obtained from Nandi et al., 2000: ctxA_For, ompW_For, ompW_Rev, ompW_Inner_Rev. 

The following primers were designed by Dr Alex Shaw and are unpublished:
ToxR_For_Outer, Tox_Rev_Outer, ToxR_For_Inner, O1_rfbFor_Outer, O1_rfbRev_Outer, O139_rfbFor_Outer, O139_rfbRev_Outer. 






[STEPS]
SECTION: Multiplex nested PCR for V.Cholera
1.  Assemble primer pool: 
 
 
Bands for the V. cholera reactions: 
 
 Reconstitute primers to 100 µM using nuclease-free water (or if primer manufacturer recommends otherwise, follow their recommendations for reconstituting primers).

 Create working stocks of 10 µM using nuclease-free water. Create 10 µM primer pools C and D by mixing together 10 µL of each primer marked as being part of the pool. Scale up as needed.  
SECTION: Multiplex nested PCR for V.Cholera
1.1. V. cholera first round PCR reaction

  

Prepare the following Master mix on ice in a 1.5ml Eppendorf Lobind tube per number of samples/controls + 10%: 

 

Briefly vortex and centrifuge down the master mix and aliquot 20 µL into each PCR tube. 
      
Add 5 µL of extracted DNA from each sample to a tube. 

Briefly vortex, and centrifuge down the PCR mixes. 
SECTION: Multiplex nested PCR for V.Cholera
1.2. Amplify using the following cycling conditions: 
 
 

SECTION: Multiplex nested PCR for V.Cholera
1.3. V. Cholera second round PCR reaction

Prepare the following Master mix on ice in a 1.5ml Eppendorf Lobind tube per number of samples/controls + 10%:
 
 
Briefly vortex and centrifuge down the master mix and aliquot 20 µL into each PCR
tube. 

Add 5 µL of first round amplicon and vortex briefly to mix. Briefly centrifuge down the PCR mixes. 
SECTION: Multiplex nested PCR for V.Cholera
1.5. Check amplicons using an agarose gel or tapestation. 
SECTION: Multiplex nested PCR for V.Cholera
1.4. Amplify using the following cycling conditions:
TITLE: Lipids annotation of Nano-DESI MSI datasets
AUTHORS: yang1832 , Julia Laskin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope: </div><div class = "text-block">Annotate lipid species detected by Nano-DESI IMS analysis.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">A list of lipid identifications with low mass errors (<5 ppm) that correlate to ion images produced by Nano-DESI IMS analysis.</div></div>

[STEPS]
?. Create mass list from the averaged spectrum.
?. Use LIPIDMAPS database to annotate each m/z value with the following search criteria:https://www.lipidmaps.org/resources/tools/bulk_structure_searches.php?database=COMP_DB1a. Positive Mode Adducts: [M+H]+, [M+Na]+, [M+K]+1b. Negative Mode Adducts: [M-H]-2.   Specify Mass Tolerance: 0.005 m/z3.   Specify Chains: Even Chains Only
?. Obtain DDA MS/MS data from each sample.
?. Use the diagnostic fragements and characteristic neutral loss to narrow down the peak assignment. Remove the assigments that does not match with the CID spectrum.
?. Calculate ppm error associated with each assignment and remove assignments with errors larger than 5 ppm, although most have errors lower than 3 ppm.
TITLE: Latkes done properly
AUTHORS: Lenny Teytelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recipe is a traditional Eastern-European Jewish latkes (potato pancakes).  This fork is my mom's recipe . It's not vegan. We eat them with spicy cranberry sauce, apples sauce and/or sour cream. But the latkes are the classic kind. </div></div>

[STEPS]
?. Do not peel!  No need to peel!  Wash 6 Russet potatoes.
?. Cut up the potatoes to large chunks. Put them in a food processor bowl. Using chopping blade in bowl process JUST until the pototoes are chopped in pieces 2 mm long (like very big rice perhaps). Some larger chunks will remain.  Do not over process. Should not be liquid.
?. Put the potatoes in a bowl and add 1 onion (chopped the same way) to the bowl.
?. Add 1 teaspoon of salt. 1/4 teaspoon baking powder, Add \frac{1}{4} cup of flour to the potatoes, 1/4 teaspoon pepper is good too. taste it, probably add more salt. Also, no one every makes one batch because they always eat the first batch. I usually start with X4 of everything.
?. Cover bottom of a frying pan with oil and heat to medium-high. Oh for heavens sakes. This is not enough oil. Add oil (we use canola but I'm always tempted to try olive as a them of the holiday) to about 3/4 inch at least. They don't absorb more if the oil is the right temperature.
?. Use a spoon to remove the water; keep doing this as you fry.Put the spoon onto the surface of the batter, pressing it down a little bit. Let the liquid seep in.   YOU DON'T HAVE TO DO THIS STEP IF YOU DON'T PROCESS TOO MUCH.
?. Drop about 2 Tablespoons in the oil. I like to form the round latke on a big spoon and slide in the oil. Fry on one side for 3-5 minutes until crisp and golden brown, but maybe still a bit soft in the middle.I can't figure out how to get my video to load so you can see how they should look! I'll get help on this!
?. Flip, add fry until both sides are golden brown. YUM
?. Now add the eggs. You can leave this out, but the texture changes.  Mix well with a spoon.
TITLE: Rapalog-induced chemical dimerization experiments
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes Rapalog-induced chemical dimerization experiments.

[STEPS]
SECTION: Rapalog-induced chemical dimerization experiments
1. To perform chemical-induced dimerization (CID) experiments we will first establish cell lines expressing the FRB-Fis1 and FKBP-GFP-GOI (FKBP-GFP fused to our gene of interest).
SECTION: Rapalog-induced chemical dimerization experiments
2. HeLa cells are consecutively transduced with lentivirus for FRB-Fis1, which also expresses mitochondrially targeted monoKeima (mt-mKeima), and then with FKBP-GFP-GOI. See the lentivirus transduction protocol for further details.

SECTION: Rapalog-induced chemical dimerization experiments
6. Analyze the cells by flow cytometry, using the mt-mKeima ratio (561/405) as a readout with an LSR Fortessa Cell Analyzer (BD Biosciences).
SECTION: Rapalog-induced chemical dimerization experiments
7. Gate the cells for GFP/mt-mKeima double-positive cells, and collect 10,000 events for this population.
SECTION: Rapalog-induced chemical dimerization experiments
8. Analyze data using FlowJo.
SECTION: Rapalog-induced chemical dimerization experiments
3. Once cell lines are established, cells are seeded in 6-well plates the day before the experiment (density: 800k per well).
SECTION: Rapalog-induced chemical dimerization experiments
4. Induce FKBP-FRB dimerisation by treating the cells with 50 nanomolar (nM) Rapalog A/C hetero-dimerizer rapalog (635057, Takara) for 1440 min.
SECTION: Rapalog-induced chemical dimerization experiments
5. After 24h, collect cells by trypsinization and take the cells to the FACS facility for mt-mKeima analysis.
TITLE: Image analysis of immediate early gene expression in spinal cord sections
AUTHORS: Janet Keast, Peregrine Osborne, Nicole Wiedmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for analysing expression pattens of immediate early gene products (e.g., c-Fos) in immunostained transverse sections of spinal cord. </div></div>

[STEPS]
?. [Creating an image mask]
Using Adobe Photoshop (or similar program) open the image of the spinal cord section to create an image mask. Create a new “layer”.
?. [Using the image mask]
Paste the mask onto the image for counting. This will add a new layer. Using the 'free transform' tool, scale and rotate the mask to align it to the image.
?. [Using the image mask]
Right click to access the the 'Warp' tool to further align the mask to the image.
?. [Using the image mask]
.Export the combined image as a TIFF.
?. [Counting]
Open the TIFF file with the overlaid mask with ImageJ (FIJI).
?. [Counting]
Set up counting: Plugins -> Analyse -> Cell Counter -> Initialize -> Add
?. [Counting]
Select the cell number that corresponds to the region being counted. Each cell number will be a different region. eg Type 1 = Laminae 1
?. [Counting]
Click on each cell in the region.
?. [Counting]
Repeat for all regions.
?. [Counting]
Save the .txt file with the xy coordinates of each cell.
?. [Using the image mask]
Open the image chosen for counting neurons and its corresponding image mask. Copy the mask by selecting the whole area of the mask.
?. [Creating an image mask]
Open the “Window” menu and select “Paths” to pull up the corresponding panel. Select the Magnetic Pen to follow transitions of color and brightness within the image. Draw your vector paths over the image until you have a traced conversion of the paths and shapes within your image. Press Enter when you are done tracing a path to signal the end of the pathway.
?. [Creating an image mask]
Set the tolerance level for the pathways. Smaller levels make the path adhere tightly to what you’ve traced, while larger levels displays smooth transitions between anchor points in your path.
?. [Creating an image mask]
Save this mask: in order to maintain a transparent background, save the pathway image as a .png file and select ‘transparency’ during exporting.
?. [3D visualization]
Completed tilescan images are then imported into TissueMaker for 3D reconstruction. Images are ordered, rotated and aligned based on structural landmarks. Reconstructed spinal cords can be visualized in a 3D space using Neurolucida 360.
TITLE: Protocol - A systematic review and meta-analysis on impact of suboptimal use of antidepressants, bisphosphonates, and statins on healthcare resource utilisation and healthcare cost
AUTHORS: Kyu  Hyung Park

[DESCRIPTION]
We conduct a systematic literature review and meta-analysis for the impact of adherence to antidepressants, bisphosphonates or statins on healthcare resource utilisation or healthcare costs, using the articles finding these impacts published between November 2004 and April 2021 searched from PubMed, Cochrane Library, JSTOR, EconLit, and ClinicalTrials.gov.

[STEPS]
TITLE: Generation of hESC/iPSC Derived Midbrain Dopamine Neurons
AUTHORS: Su-Chun Zhang

[DESCRIPTION]
This protocol details methods for generating Midbrain Dopamine Neuronds from hESC or iPSC sources. 

[BEFORE_START]
Induction of the midbrain dopaminergic progenitors was carried out based on protocol described previously (Xi et al., 2012) with modification. 


[GUIDELINES]
References
 

 


[STEPS]
SECTION: Pre-differentiation and Differentiation (Day 1-6)
1. Briefly, culture hESCs (1 day after passaging) on MEF feeder layer in the neural induction medium (NIM) (DMEM/F-12, 1X NEAA, 1X N2 supplement) supplemented with 10 micromolar (µM) and 2 micromolar (µM). 

To pattern the differentiating cells to the midbrain FP progenitors, add  500 nanogram per milliliter (ng/mL) and 0.4 micromolar (µM) to the cultures from day 1 till day 7.

SECTION: Differentiation (Day 7-11)
2. On day 7, gently blow off individual colonies of neuroepithelial cells with a pipette and replate on mouse embryonic fibroblast feeder in the NIM containing 1 micromolar (µM) and 100 nanogram per milliliter (ng/mL) and 0.4 micromolar (µM) for additional 6 days (D7-12).

SECTION: Differentiation (Day 12-19)
3. On day 12, remove CHIR99021, reduce the following:  20 nanogram per milliliter (ng/mL), 0.5 micromolar (µM) and 100 nanogram per milliliter (ng/mL) and add to the culture to expand the progenitors in suspension till day 19.

SECTION: Differentiation (Day 19-31)
4. Then, keep 20 nanogram per milliliter (ng/mL) and 20 nanogram per milliliter (ng/mL)  in the neural induction medium till transplantation/in vitro analysis at day 32.

SECTION: In Vitro Analysis (Day 32+)
5. For in vitro analysis, dissociate neurospheres by incubation in Accutase at 37 °C for 3 min-5 min on day 32 and plate onto glass coverslips that were previously coated with Matrigel.

SECTION: In Vitro Analysis (Day 32+)
6. Feed cells with Neural differentiation medium (NeurobasalTM Medium, 1 X N2 supplement, 1 X B27 supplement) (NDM) supplemented with brain-derived neurotrophic factor (10 nanogram per milliliter (ng/mL)), glial cell line derived neurotrophic factor (10 nanogram per milliliter (ng/mL)), ascorbic acid (200 micromolar (µM)), 1 micromolar (µM), transforming growth factor β3 (1 nanogram per milliliter (ng/mL)) and Compound E (0.1 micromolar (µM)).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay </strong></p>
<p> </p>
<p>Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize        2 ʹ, 3 ʹ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2ʹ-5ʹ and 3ʹ-5ʹ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (K<sub>D </sub>= 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.</p>

[STEPS]
TITLE: Direct ELISA
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol details Direct ELISA.

[STEPS]
SECTION: Direct ELISA
1. Dilute 100 ng - 500 ng of protein of interest (e.g. α-synuclein) in Takeda buffer per well and add 30 µL total liquid per well to 384-well Nunc Maxisorp plate.

SECTION: Direct ELISA
2. Seal with removable clear adhesive cover.
SECTION: Direct ELISA
3. Centrifuge the plate at 1000 x g for 1 min to pull down protein onto the plate. Leave for
240 min at 37 °C or   at 4 °C.

SECTION: Direct ELISA
4. Use the plate washer with 5x with 100 µL PBST.
SECTION: Direct ELISA
5. Block with 100 µL Blockace per well. Fill wells from the bottom, being sure to avoid
leaving any bubbles in the wells.

SECTION: Direct ELISA
6. Seal with a removable clear adhesive cover and leave for 240 min at 37 °C or  at 4 °C.

 




SECTION: Direct ELISA
7. Use the plate washer with 5x with 100 µL PBST.

SECTION: Direct ELISA
8. Use C buffer to dilute reporter antibody. Vortex immediately before pipetting. 
SECTION: Direct ELISA
9. Using multichannel, fill 91, dispense 30 µL three times.

SECTION: Direct ELISA
10. Seal with removable clear adhesive cover and centrifuge plate at 1000 x g for 1 min.

SECTION: Direct ELISA
11. Incubate for 240 min at 37 °C or   at 4 °C.

SECTION: Direct ELISA
12. Use C buffer to dilute HRP-conjugated secondary reporter antibody.
SECTION: Direct ELISA
13. Add 30 µL per well. For goat-anti-mouse/rabbit use at 1:5-20K.

SECTION: Direct ELISA
14. Seal with removable clear adhesive cover and centrifuge plate at 1000 x g for 1 min.

SECTION: Direct ELISA
15. Incubate for 60 min at 37 °C.

SECTION: Direct ELISA
16. Use the plate washer with 5x with 100 µL PBST.

SECTION: Direct ELISA
17. Add 30 µL TMB reagent per well.

SECTION: Direct ELISA
18. Develop for 10 - 30 min. 

SECTION: Direct ELISA
19. Quench using 30 µL 10% phosphoric acid per well.

SECTION: Direct ELISA
20. Read plate on the Spectramax or similar plate reader. 384-495 nm for unquenched
reactions, 450 nm for quenched reactions.
TITLE: Stereology-mediated cell count using StereoInvestigator
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
Protocol for cell counting using StereoInvestigator software. 


[STEPS]
1. Set slide properly on the microscope and set new reference point
2. Click on Probes → Optical Fractionator Workflow → Start a new subject  →
3. Enter cut thickness: 30 µm (cut with cryostat)
4. Enter interval according to the thickness: 5 if SNpc, 6 if STR 
5. In Select Low Mag Lens, click 5X
5.1. Click on Next Step
6. Select the Contour of Interest(s)
7. In Select High Mag Lens select 40X
7.1. Click on Next Step
8. In Manually enter the average mounted thickness enter the corresponding one
8.1. Click on Next Step
9. In Define the Counting frame enter: 80 x 80 µm
9.1. Click on Next Step
10. Enter 25 Percentage
10.1. Click on Display Changes
11. Enter: 240x200 µm if SNpc, 620 x 490 µm if STR 
11.1. Click on Display Changes
12. In Optical Disector Height enter: 5 µm, 20 µm 
11.2. Click on Next Step
12.1. Click on Next Step
13. Select your first section
13.1. Click on Yes
14. Select the desired region
14.1. Click on start counting
15. Focus on the tissue
15.1. Click on OK
16. Select a number of markers according to the desired cell types 
 

17. Count every defined region from every set section 
17.1. Click on Next Step
17.2. Click on Display Probe Run List
18. Click on Section Name → Click on Counter/Marker Name → Select all sections belonging to the same region
19. Click on View Results
 

20. Click on the different markers to see the number of cells counted and the estimated cell number
20.1. Click on CE Scheaffer to view the area if needed
20.2. Repeat steps 17 through 20.1 for each region