eamag/protocols-with-steps huggingface dataset:
TITLE: Cathepsin D assay to verify the retention of lysosomal content
AUTHORS: Rotimi Y. Fasimoye, Dario R. Alessi

[DESCRIPTION]
Cathepsin D assay is a fluorescence-based assay that leverage on the activity of cathepsin D, a lysosomal enzyme, to monitor the intactness of lysosome in the cell. Here, we describe a method where we used the measurement of cathepsin D activity to verify the intactness of lysosomes that were isolated from HEK293 cells based on anti-TMEM192 Lyso-IP. Our data showed an increase in the cathepsin D activity of lysosomal fraction when compared with whole cell fraction and Mock-IP fraction, an indication that the lysosomes are intact and viable.

[STEPS]
SECTION: Seeding cells and performing Lyso-IP with anti-TMEM192 beads
1. Seed HEK293 cells in 15cm plates and allow to reach 80-90% confluency.
SECTION: Seeding cells and performing Lyso-IP with anti-TMEM192 beads
2. Perform Lyso-IP (using anti-TMEM192 beads) and Mock-IP (using BSA coated beads) as previously described in dx.doi.org/10.17504/protocols.io.x54v9yp51g3e/v1
SECTION: Preparing sample for Cathepsin D assay
3. Add 2 µg of protein from Lyso-IP and whole cell lysate into the wells FLUOTAC flat bottom black 96-well plate. This should be done in duplicate.

SECTION: Preparing sample for Cathepsin D assay
4. Top up to 50 µLwith lysis buffer provided in the kit.


 


SECTION: Preparing sample for Cathepsin D assay
5. Prepare Blank sample in duplicate. This should contain only lysis buffer.
SECTION: Preparing sample for Cathepsin D assay
6. Prepare a reaction master mix for 9 wells:

450 µL reaction buffer (from the kit) per well.
18 µL substrate (from the kit)


 

SECTION: Preparing sample for Cathepsin D assay
7. Add 52 µL of master mix into each well. Gently mix but avoid bubbles. Cover plate with foil to avoid light exposure.

SECTION: Plate reading and analysis.
8. Start the ClarioStar plate reading machine and initiate the software. 
SECTION: Plate reading and analysis.
9. Set temperature to 37 °C.

SECTION: Plate reading and analysis.
10. Set reading time to 5 min for 24 cycles. This is total reading time of 120 min.

SECTION: Plate reading and analysis.
11. Set reading wavelength to Ex/Em =328 1535 / 460 1535 .

SECTION: Plate reading and analysis.
12. Highlight and name the virtual wells, ensuring they correspond with the orientation of the plate.
SECTION: Plate reading and analysis.
13. Set direction of plate reading. 
SECTION: Plate reading and analysis.
14. Open the plate holder and insert plate. Remember to remove foil covering before inserting the plate into the equipment.
SECTION: Plate reading and analysis.
15. Close plate holder and run the program.
SECTION: Plate reading and analysis.
16. After the completion of the run, export data in excel format and analyse it using GraphPad Prism.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes isolation of PBMC from human whole peripheral blood samples by density gradient centrifugation, using One SepMate-50ml tube (<a href="https://www.stemcell.com/sepmate-trade-hassle-free-pbmcs-in-just-15-minutes.html" target="_blank">https://www.stemcell.com/sepmate-trade-hassle-free-pbmcs-in-just-15-minutes.html</a>), followed by freeze and thaw protocol.</p>

[STEPS]
TITLE: Chile atole verde - test for draft posting
AUTHORS: Gabriel  Gasque

[DESCRIPTION]
Test for draft posting

[STEPS]
SECTION: Salsa verde
1. Asa el chile poblano y los chiles jalapeños. No es necesario pelar los chiles.
 

SECTION: Chileatole
5. Sofríe la cebolla por 3 min o hasta que esté translúcida y sin dorarse. 

SECTION: Chileatole
6. Agrega el ajo y cocina por 1 min.

SECTION: Salsa verde
2. Licúa los chiles asados con la espinaca, la hoja santa y las 6 hojas de epazote en 1 taza de agua hasta obtener una sala tersa.
 

SECTION: Chileatole
7. Agrega el caldo de verduras o pollo, los granos de elote, y el elote troceado y deja hervir a fuego bajo (simmer) por 20 min hasta que el elote este tierno.

SECTION: Chileatole
4. Disuelve la masa de maíz (1/4 de taza) en 1 taza de caldo frio de verduras (o de pollo o agua). Reserva
SECTION: Salsa verde
3. Prueba de sal y ajusta. 
SECTION: Chileatole
8. Agrega la salsa verde y la masa de maíz previamente disuelta en el agua.
SECTION: Chileatole
9. Deja hervir a fuego lento (simmer) por 10 min moviendo constantemente para que no se pegue.

SECTION: Servir
11. Sirve el chileatole caliente y acompáñalo de queso fresco desmenusado, cebolla morada picada, epazote y una rodaja de limón.
SECTION: Chileatole
10. Ajusta la sal.
TITLE: LC-MS of Native Nanodiscs
AUTHORS: Caroline Brown, Snehasish Ghosh, Kallol Gupta

[DESCRIPTION]
This protocol provides a step-by-step guide for conducting LC-MS analysis of native nanodiscs extracted from biological membranes.

[STEPS]
SECTION: Liquid Chromatography
1. Resuspend peptides in water + 0.1 formic acid varying amount of water so that 500ng of peptide can be loaded in a reasonable volume.
SECTION: Liquid Chromatography
2. Chromatography is subsequently conducted using home-packed C18 columns (15cm x 75uM ID) for separation of peptides by hydrophobicity.
SECTION: Liquid Chromatography
3. Use a 75 minute separation gradient with a buffer system of water/acetonitrile with 0.1% formic acid beginning at 6% acetonitrile and ending at 100% acetonitrile.
SECTION: Mass Spectrometry
4. Data Dependent Acquisition (DDA) mass spectrometry is conducted using an Orbitrap Eclipse instrument. 
SECTION: Mass Spectrometry
5. MS1 scans a acquired using the Orbitrap detector at a resolution of 120000 and stored in profile mode using a mass range of 350-1400 m/z and and intensity threshold of 1.0e3.
SECTION: Mass Spectrometry
6. Use a cycle time of 1 second for ion isolation and MS2 fragmentation isolating ions with a 1.4 m/z window and fragmented with CID energy at 30% activation. 
SECTION: Mass Spectrometry
7. Product ions are detected at the ion trap set to rapid detection mode with precursors ions being dynamically excluded for 20 seconds after one instance of detection.
TITLE: Symbiont density quantification in live Aiptasia
AUTHORS: Shumpei  Maruyama, OSU Weis Lab, Virginia Weis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed for quantifying symbiont density during the onset of
symbiosis and early proliferation. Other techniques should be used for quantifying high symbiont density.</div></div>

[STEPS]
?. [Anemone preparation]
After initial inoculation, re-plate anemones in a clean plate to avoid imaging algae that are not inside the host.
?. [Imaging]
Remove half of the seawater from each well.
?. Add MgCl2 (3.76g MgCl2 in 50mL filtered seawater) to well for a 1:1 dilution.
Note: start with one anemone at a time so you don’t overstress them, but as you get quicker you can move up to 2-3 anemones at a time.
?. Wait at least 10 minutes for full relaxation. If tentacles move during imaging, subsequent image processing will be difficult, so make sure tentacles are not moving excessively and you may need to wait longer than 10 minutes for full relaxation.
?. Spread out the anemone tentacles by pipetting the MgCl2 + seawater solution repeatedly. In the relaxation solution, the tentacles will shrink, so forcing a flow of water to the anemone expands the tentacles.
?. Remove ~75% of the solution from the well so that the tentacles are laid relatively flat.
?. Take care that the tentacles are not directly on the bottom of the plate to avoid imaging free algae from bottom of well.
?. Take photos at the base of tentacles near oral disk in brightfield (multiple focal points if needed) and of symbionts with green laser/red filter (again at multiple focal points).
?. Repeat two more times for a total of 3 tentacles per anemone.
?. Some movement of the tentacles is tolerated by the image processing, but excess movement will lead to duplication of algal counts. Be mindful of drift.
?. The whole process should take about 20-30+ minutes (from step 1), do not leave anemones in relaxation solution longer than that. Repeat until all anemones are imaged.Tip: As you begin imaging an anemone, you can add MgCl2 to the next 1-3 anemones so that you do not need to wait 10 minutes between anemones. They will be ready to image by the time you are done imaging your current anemone.
?. [Image processing]
Using Adobe Photoshop and the provided macro, focus stack brightfield photos and algal photos separately for each tentacle. You should get one image for brightfield and one image of algal fluorescence.
?. Using ImageJ and the provided macro, hand-select tentacle area with the bf image, and manually adjust threshold of symbiont fluorescence picture to obtain symbiont count and mm2 tentacle area. You can then calculate symbionts/mm2 per tentacle.The provided macro includes a calibration for pixels to mm length, so please edit this parameter to meet your needs.
TITLE: TXTL phage production
AUTHORS: Marijn Ceelen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains how to use the Abor BiosciencesSigma 70 TXTL mix to produce bacteriophages. This protocol works for bacteriophage T7. It has been tested on phage lambda. However, it seems that the current protocol does not work for phage lambda. </div></div>

[STEPS]
?. Calculate ratios of ingredients. TXTL master mix should always be ¾ of the reaction.Concentrations of other ingredients can be varied, but these are the tested concentrations:  AB1Ingredient&nbsp;Concentration&nbsp;2PEG-8000&nbsp;3%&nbsp;3GamS&nbsp;3.3&nbsp;µM&nbsp;4dNTPs&nbsp;0.5&nbsp;mM&nbsp;The remaining part of the reaction should consist of the DNA of choice and MQ water.As a positive control for the TXTL reaction GFP can be used. Make sure that the GFP plasmid has a sigma 70 promoter and that the sample is RNase free. For the Arbor Biosciences GFP positive control plasmid the recommended concentration is 5 nM.A reaction volume of 12 µL is recommended by Arbor Biosciences. Reactions can be carried out in 96 wells plates or in eppendorfs. When using a 96 wells plate, make sure to seal the plate properly to prevent contamination.  1 Rustad, M., Eastlund, A., Jardine, P., & Noireaux, V. (2018). Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synthetic Biology, 3(1), ysy002.
AB1Ingredient&nbsp;Concentration&nbsp;2PEG-8000&nbsp;3%&nbsp;3GamS&nbsp;3.3&nbsp;µM&nbsp;4dNTPs&nbsp;0.5&nbsp;mM&nbsp;
Concentration of DNA used can be varied. Recommended DNA concentration according to literature is 0.25 nM of DNA for phage T7. [1] Lower DNA concentrations do also work. T7 phage production has been observed at concentrations as low as 0.04 nM. For different phages, different genome concentrations might be optimal.
?. After pipetting the ingredients together, the mixture should be incubated for  to  at . Other temperatures might be optimal for different phages.
30 °C
?. To check for phage production, a plaque assay can be done.
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TITLE: Data for manuscript: Estimation of seed yield in oilseed rape to identify the potential of semi-resynthesized parents for the development of new hybrid cultivars
AUTHORS: Laurencja Szała, Zygmunt Kaczmarek, Wiesława Popławska, Alina Liersch, Marek Wójtowicz, Marcin Matuszczak, Zdzisław R. Biliński, Katarzyna Sosnowska, Michał Stefanowicz, Teresa Cegielska-Taras

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Resynthesized (RS) </span><span style = "font-style:italic;">Brassica napus</span><span> can be used to increase the genetic diversity of this important crop plant and to develop the heterotic gene pool required for successful hybrid breeding programmes. The level of heterosis in F</span><span style = "vertical-align:sub;">1</span><span> hybrids depends on the individual performance of the parents and on the degree of genetic difference between them. However, RS forms obtained from crosses of </span><span style = "font-style:italic;">B. rapa</span><span> ssp. with </span><span style = "font-style:italic;">B. oleracea</span><span> ssp. possess many undesirable agronomic traits, such as low quality of seeds, low yield and seed oil content, high erucic acid level in the oil and high glucosinolate content in seed meal. Therefore, RS oilseed rape needs to be improved by crossing with natural double-low oilseed rape, leading to selected double-low quality semi-RS lines that can be used for breeding. In this study, we evaluated the seed yield potential of F</span><span style = "vertical-align:sub;">1</span><span> hybrids derived from crosses between Ogura cytoplasmic male-sterility (CMS) lines and doubled haploid (DH) semi-RS restorer lines in three locations in Poland. The genotype by environment interaction (GE interaction) and general combining ability (GCA) of the restorer and CMS line effects, as well as the effects of heterosis, were also assessed. The results of the study provide the first insights into the use of semi-RS lines as components for the development of new hybrid cultivars. Even the introduction of 50% of the RS oilseed rape genotype to natural restorer lines resulted in a marked heterosis effect, with seed yield ranging from 4.56% to 90.17% more than that of the better parent. The yield of the best hybrid amounted to 108.6% of the seed yield of the open-pollinated cultivar Monolit and 94.4% of that of the hybrid cultivar Arsenal. The best DH semi-RS line, S1, which had a significantly positive GCA for seed yield, can be recommended as a possible parent for inclusion in breeding programmes aimed at developing new hybrid cultivars.</span></div><style>
							  .justify:after {
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							    display:inline-block;
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							</style></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Case 2: Fix samples. Use this protocol when long term storage of slide is required.<br /><br />For when you can count your samples in a few days, see <a href="https://www.protocols.io/view/SYBR-Gold-Staining-for-Viral-Enumeration-Case-1-c69zh5" target="_blank">Case 1</a>.&nbsp;

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: E. coli K12 DNA Extraction
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>How to extract DNA from </span><span style = "font-style:italic;">E. coli</span><span> K12 using Wizard® Genomic DNA Purification Kit by Promega®.</span></div><div class = "text-block"><span>I do not claim any credit for the development of this protocol. It has been adapted from the protocol detailed in: </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Culture bacteria]
Culture E. coli K12 in BHI broth overnight.lyophilized E. coli K12 in BHI broth.
2 mg
10 ml
?. [Pellet the cells]
Add cell suspension to 2 mL microcentrifuge tube.
1 ml
?. [Pellet the cells]
Centrifuge at 13,000-16,000 × g for .
?. [Pellet the cells]
Remove supernatant.
?. [Lyse nuclei]
Add of Nuclei Lysis Solution.
600 µl
Nuclei Lysis Solution is marked "NL"
?. [Lyse nuclei]
Gently pipet until the cells are resuspended.
?. [Lyse nuclei]
Incubate aton heating block for to lyse the cells.
?. [Lyse nuclei]
Cool to room temperature.
?. [Precipitate proteins]
Add of Protein Precipitation Solution to the RNase-treated cell lysate.
200 µl
Protein Precipitation solution is marked "P"
?. [Precipitate proteins]
Vortex vigorously at high speed for .
?. [Degrade RNA]
Add RNase Solution to the cell lysate.
3 µl
RNAse solution is marked "R"
?. [Degrade RNA]
Invert 2-5 times to mix.
?. [Degrade RNA]
Incubate at for to .
?. [Precipitate proteins]
Incubate on ice for
?. [Degrade RNA]
Cool to room temperature.
?. [Harvest DNA]
Transfer the supernatant containing the DNA to a clean 1.5 mL microcentrifuge tube containing isopropanol.
600 µl
Some supernatant may remain in the original tube conatining the protein pellet. Leave this residual to avoid contaminating the DNA solution with the precipitated protein.
Isopropanol is marked "IPA"
?. [Precipitate proteins]
Centrifuge at 13,000-16,000 × g for .
?. [Harvest DNA]
Gently mix by inversion until the thread-like strands of DNA form a visible mass.
?. [Wash and dry DNA]
Centrifuge at 13,000-16,000 × g for .
?. [Wash and dry DNA]
Carefully pour off the supernatant and drain the tube on clean absorbent paper.
?. [Wash and dry DNA]
Add of 70% ethanol and gently invert the tube several times to wash the DNA pellet.
600 µl
70% ethanol solution is marked "Et"
?. [Wash and dry DNA]
Centrifuge at 13,000-16,000 × g for .
?. [Wash and dry DNA]
Carefully pour off the ethanol.
?. [Wash and dry DNA]
Drain the tube on clean absorbent paper and allow to air-dry for 10-15 minutes.
?. [Rehydrate DNA]
Add of DNA rehydration solution to the tube.
100 µl
DNA Rehydration solution is marked "DR"
?. [Rehydrate DNA]
Rehydrate by incubating the solution overnight at room temperature or .
?. [Rehydrate DNA]
Store DNA at to .
?. [Pellet the cells]
Label centrifuge tube with your group number.
?. [Harvest DNA]
Label centrifuge tube with your group number.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Provide a modified Bradford method to determine the residual protein content in the bio-synthesis product. </p>
<p>The product may be obtained from enzymatic reaction and extract from organic solvent, and /or  insoluble in water.</p>

[GUIDELINES]
<p>May not suitable the product wo dissolved in some organic reagents/ water buffer, need to test.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Rat astrocyte isolation and culture
AUTHORS: Shiyi Wang

[DESCRIPTION]
Rat astrocyte isolation and culture

[STEPS]
1. **Cortex Dissection and Digestion** - Micro-dissect P1 rat cortices from both sexes. - Digest cortices in papain solution.
2. **Trituration and Resuspension** - Triturate tissue in low and high ovomucoid solutions. - Resuspend cells in astrocyte growth media (AGM): - DMEM (GIBCO 11960) - 10% FBS - 10 μM hydrocortisone - 100 U/ml Pen/Strep - 2 mM L-Glutamine - 5 μg/ml Insulin - 1 mM Na Pyruvate - 5 μg/ml N-Acetyl-L-cysteine
3. **Cell Plating and Incubation** - Plate 15-20 million cells on 75 mm² flasks (non-ventilated cap) coated with poly-D-lysine. - Incubate at 37°C in 10% CO2.
4. **Non-Astrocyte Cell Removal (DIV 3)** - Forcefully shake closed flasks by hand for 10-15 seconds to remove non-astrocyte cells. - Ensure only an adherent monolayer of astrocytes remains.
5. **AraC Treatment (DIV 5 to DIV 7)** - Add AraC to the media to eliminate contaminating fibroblasts.
6. **Astrocyte Passage and Plating (DIV 7)** - Trypsinize astrocytes with 0.05% Trypsin-EDTA. - Plate cells into 12-well or 6-well dishes.
7. **Transfection of Astrocytes (DIV 8)** - Transfect with shRNA and/or expression plasmids using Lipofectamine LTX with Plus Reagent (Thermo Scientific). - Dilute 1 μg (12-well) or 2 μg (6-well) total DNA in Opti-MEM with Plus Reagent. - Mix with Opti-MEM containing LTX (1:2 DNA to LTX ratio). - Incubate for 30 minutes at room temperature. - Add transfection solution to astrocyte cultures and incubate at 37°C for 3 hours.
8. **Co-Culture with Neurons (DIV 10)** - Trypsinize astrocytes on DIV 10. - Resuspend in NGM Plus. - Plate 20,000 cells per well onto DIV 10 neurons. - Co-culture for 48 hours.
TITLE: darkmode protocol
AUTHORS: Maria Gul

[DESCRIPTION]
Piqued favour stairs it enable exeter as seeing. Remainder met improving but engrossed sincerity age. Better but length gay denied abroad are. Attachment astonished to on appearance imprudence so collecting in excellence. Tiled way blind lived whose new. The for fully had she there leave merit enjoy forth.

Betrayed cheerful declared end and. Questions we additions is extremely incommode. Next half add call them eat face. Age lived smile six defer bed their few. Had admitting concluded too behaviour him she. Of death to or to being other.

[STEPS]
1.  
 
 

Piqued favour stairs it enable exeter as seeing. Remainder met improving but engrossed sincerity age. Better but length gay denied abroad are. Attachment astonished to on appearance imprudence so collecting in excellence. Tiled way blind lived whose new. The for fully had she there leave merit enjoy forth.

Betrayed cheerful declared end and. Questions we additions is extremely incommode. Next half add call them eat face. Age lived smile six defer bed their few. Had admitting concluded too behaviour him she. Of death to or to being other.
TITLE: Protocol 3: Antiobiotic Resistance
AUTHORS:  

[STEPS]
?. Plate the CRISPR solution. Take LB  Strep/Kan/Arabinose agar plate out of the fridge and let sit to adjust to room temperature. Shake the microcentrifuge tube containing the CRISPR experiment and then pour or pipette contents onto the agar plate. Use an inoculation loop to spread contents onto the plate. Let dry for a few minutes. Only let the plate dry in a sanitary space or other contaminents may join the plate.
?. Incubate contents on LB plate. Put the lid back on, flip the plate over, make sure to label it, and leave the bacteria to culture in room temperature. It will take 1 to 2 days to grow.
?. Congratulations! You have now completed your first CRISPR experiment! Go you.
?. Take a picture of your end culture and input it on your Lab Notebook. If you did not see any growth, identify factors that may have contributed to this result.
TITLE: Protocol for Nuclei/ Cell Isolation and 10X Genomics Fixed RNA profiling for Human Ovary
AUTHORS: Nicolas Martin

[DESCRIPTION]
This is the 10X Genomics protocol to fix, dissociate, and profile RNA from human ovary tissue. 

[GUIDELINES]
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Cell/Nuclei Isolation Protocol for Human Skeletal Muscle
1. The protocol CG000553 REV B was used to fix, dissociate, and isolate cells/nuclei from frozen human skeletal muscle with the following modifications: 

1) 0.25 mg / mL of Liberase TH was used for dissociation at Step 2b, Page 6.
2) Two extra "spin only" (i.e., steps 3 and 4 ) of the Octodissociator protocol were run for each sample due to the presence of intact/ large tissue pieces at the end of the run at Step 2c, Page 6.   
3) Counts were performed using the Cellaca PLX Automated Cell counter (PN: PLX-SYS1) and ViaStain AOPI staining solution (PN: CS2-0106-5mL) at Step 2i on page 6. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/sample-prep/tissue-fixation-and-dissociation-for-chromium-single-cell-gene-expression-flex


SECTION: Chromium Fixed RNA Profiling Reagent Kits
2. The protocol CG000527 Rev E  was used to generate gene expression libraries from fixed cell/nuclei suspension inputs.

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples

Samples were multiplexed in batches of 16 using the user guide CG000565. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples
TITLE: Total Flavonoid Content (TFC)
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The amounts of total flavonoids was quantified. The reaction mixture consisted of 1.0 ml of extract, 0.3 ml of 5% sodium nitrite and 4 ml of 60% ethanol. After 6 min, 0.3 ml of 10% aluminium nitrite was added. In the next 6 minutes, 4 ml of 1 M sodium hydroxide solution were added. Then, the volumetric tube was diluted to 10 ml. The absorbance was measured at 510 nm.TFC was calculated and expressed as rutin equivalents (RE). A calibration curve was constructed with different concentrations of rutin (15–75 mg/ml)as a standard.</div></div>

[STEPS]