eamag/protocols-with-steps huggingface dataset:
TITLE: Immunohistochemistry against p62 and LBP110
AUTHORS: Dennis de Bakker

[DESCRIPTION]
Immunohistochemistry staining co-labeling LBP110 and p62/SQSTM1 and DAPI. 

[STEPS]
SECTION: Tissue preparation
1. Day 1: Whole mouse brain (2 years old, male) was fixed overnight in 4%PFA. 
SECTION: Tissue preparation
2. Day 2: Brain was washed 3x 5' in PBS + 4% Sucrose

SECTION: Tissue preparation
3. Day2: Brain was incubated overnight in PBS + 30% Sucrose
SECTION: Tissue preparation
4. Day3: Brain was embedded into optimal cutting medium (OCT)
SECTION: Tissue preparation
5. Day3: Brain was cut with a cryostat in 10um thick sections and stored at -80C
SECTION: Antibody staining protocol day 1
6. Slides with brain tissue sections were thawed 15 min Room temperature 

SECTION: Antibody staining protocol day 1
7. Add slides to glass coplin Jar.
SECTION: Antibody staining protocol day 1
8. Wash 3x 10 min in PEMTx

SECTION: Antibody staining protocol day 1
9. Move slides to plastic coplin jar for antigen retrieval
SECTION: Antibody staining protocol day 1
10. Perform antigen retrieval 15 min 85 °C 

SECTION: Antibody staining protocol day 1
11. Let plastic coplin jar cool down to room temperature 20 min 

SECTION: Antibody staining protocol day 1
12. Place slides back in original glass complin jar
SECTION: Antibody staining protocol day 1
13. Wash 2x 10 min in PEMTx

SECTION: Antibody staining protocol day 1
14. Place slides horizontally in box humidified with MQ. Use hydrophobic pen to outline tissue sections.
SECTION: Antibody staining protocol day 1
15. Add blocking buffer to the slides and incubate 60 min 

SECTION: Antibody staining protocol day 1
16. Add antibody solution (blocking buffer + 1:100 LBP110 + 1:100 p62) and incubate   4 °C 

SECTION: Antibody staining protocol day 2
17. Wash slides 4x 15 min 

SECTION: Antibody staining protocol day 2
18. Incubate with secondary antibody solution in humidified box (PEMTx + 2% BSA + 1:500 secondary antibodies + 1:1000 DAPI) for 60 min

SECTION: Antibody staining protocol day 2
19. Place slides back in coplin jar (keep in dark)
SECTION: Antibody staining protocol day 2
20. Wash 3x 10 min in PEMTx

SECTION: Antibody staining protocol day 2
21. Wash 2x 7 min in PEM

SECTION: Antibody staining protocol day 2
22. Mount slides using Prolong Gold mounting medium and store at 4 °C
TITLE: 1. Sample_prep_WB
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sample preparation for Western bolt assay</div></div>

[STEPS]
?. [Sample preparation for Western bolt assay]
Grow cells, harvest, wash with DI water, and store cell pellet at -800C
?. [Sample preparation for Western bolt assay]
Lysis buffer(50 mM Tris HCl pH 7.5, 200 mM NaCl, 5% Glycerol)- we don’t add DTT or PMSF(https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/index.html)    For 100 ml        a. 1M TrisHClpH 7.55 ml        b. 5MNaCl4 ml        c. 80% Glycerol6.25 ml        d. Distilled waterupto100 mlBefore use, add 1 tablet of a Protease inhibitor to 10 ml lysis buffer.After mixing with cells, add 300ug/ml final concentration lysozyme and let it sit on ice for 1-4 hrs.
?. [Sample preparation for Western bolt assay]
Bead beating and separation of Cytoplasmic and Membrane fractiona. Follow the“Bead Beating” protocolb. Centrifuge high speed for 30 minutes to get supernatant (cytoplasmic) and pellet (membrane fraction)If required to separate inner and outer membrane follow “Subcellular fractionation protocol”.If required perform acetone precipitation of the protein following “Acetone precipitation protocol”.
?. [Sample preparation for Western bolt assay]
Use the“Bradford assay” protocol to measure protein concentration and mix your protein sample with 2X Tricine sample buffer such that you have 0.5-1ug/ul of protein.
?. [Sample preparation for Western bolt assay]
2X Tricine Sample buffer(Novex, 2003)450mMTrisHCl12% Glycerol  4% SDS  0.0025% Coomassie Blue G  0.0025% Phenol Red  pH 8.45
TITLE: Homemade Gibson Mastermix
AUTHORS: Anna Behle, Tatsuya Sakaguchi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Recipe for homemade 2 x Gibson Mastermix. </div></div>

[STEPS]
?. [Preparation of 5x isothermal reaction buffer]
Recipe for 4 mL:  ABCD1ComponentMolarity / ConcentrationAmountFinal concentration2Tris-HCl, pH 7.51 M2 mL500 mM3MgCl21 M200 µL50 mM4dATP100 mM40 µL1 mM5dCTP100 mM40 µL1 mM6dGTP100 mM40 µL1 mM7dTTP100 mM40 µL1 mM8DTT1 M200 µL50 mM9PEG-8000-1 g25 %10NAD+100 mM200 µL5 mM11H2O-to final volume of 4 mL&nbsp;
ABCD1ComponentMolarity / ConcentrationAmountFinal concentration2Tris-HCl, pH 7.51 M2 mL500 mM3MgCl21 M200 µL50 mM4dATP100 mM40 µL1 mM5dCTP100 mM40 µL1 mM6dGTP100 mM40 µL1 mM7dTTP100 mM40 µL1 mM8DTT1 M200 µL50 mM9PEG-8000-1 g25 %10NAD+100 mM200 µL5 mM11H2O-to final volume of 4 mL&nbsp;
?. [Preparation of 5x isothermal reaction buffer]
Mix dNTPs, NAD+, Tris-HCl, MgCl2 and DTT.
?. [Preparation of 5x isothermal reaction buffer]
Slowly add PEG-8000 to mixture and mix well, until completely dissolved. Add H2O to a final volume of 4 mL.
?. [Preparation of 5x isothermal reaction buffer]
Prepare aliquots of the 5x isothermal buffer as required, e.g. 100 µL.Store at -20 ˚C.
?. [Preparation of 1,33x Assembly Mastermix]
Recipe for 25 x 15 µL aliquots:  ABCD1&nbsp;Component&nbsp;Concentration&nbsp;Amount&nbsp;Final concentration (after adding DNA)2&nbsp;5x isothermal rxn buffer&nbsp;5x&nbsp;80 µL&nbsp;1x3&nbsp;Taq DNA Ligase&nbsp;40 U/µL&nbsp;40 µL&nbsp;4 U/µL&nbsp;4&nbsp;T5 Exonuclease&nbsp;1 U/µL&nbsp;1.6 µL&nbsp;4 U/mL5&nbsp;Phusion High-Fidelity DNA Polymerase&nbsp;2 U/µL&nbsp;5 µL&nbsp;25 U/mL6&nbsp;H2O&nbsp;&nbsp;173.4 µL (to 300 µL)&nbsp;  ABCD1&nbsp;Total volume&nbsp;300 µL&nbsp;
ABCD1&nbsp;Component&nbsp;Concentration&nbsp;Amount&nbsp;Final concentration (after adding DNA)2&nbsp;5x isothermal rxn buffer&nbsp;5x&nbsp;80 µL&nbsp;1x3&nbsp;Taq DNA Ligase&nbsp;40 U/µL&nbsp;40 µL&nbsp;4 U/µL&nbsp;4&nbsp;T5 Exonuclease&nbsp;1 U/µL&nbsp;1.6 µL&nbsp;4 U/mL5&nbsp;Phusion High-Fidelity DNA Polymerase&nbsp;2 U/µL&nbsp;5 µL&nbsp;25 U/mL6&nbsp;H2O&nbsp;&nbsp;173.4 µL (to 300 µL)&nbsp;
ABCD1&nbsp;Total volume&nbsp;300 µL&nbsp;
?. [Preparation of 1.33x Assembly Mastermix]
Work on ice. Mix H2O and 5x buffer, then add enzymes.
?. [Preparation of 1.33x Assembly Mastermix]
Prepare 20 x 15 µL aliquots in PCR tubes. Store at -20 ˚C.These aliquots are concentrated 1.33 x - add your DNA in a volume of 5 µL to a final volume/concentration of 20 µL / 1x.
?. [Gibson assembly]
After addition of DNA, incubate Gibson assembly mix at 50 ˚C for 2 hours.
[Gibson assembly]
?. [Transformation]
Transform chemically competent cells with an aliquot of your assembly mix.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for Immunohistochemistry free-floating sections with anti-NeuN antibody for avian tissue.</p>

[STEPS]
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TITLE: αS PFF Clearance Assay Protocol
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the clearance of αS PFF assay.

[STEPS]
SECTION: αS PFF Clearance Assay Protocol
1. Prepare S PFF working solution as above (for 4 1527 final concentration).

SECTION: αS PFF Clearance Assay Protocol
2. Treat for 120 min.

SECTION: αS PFF Clearance Assay Protocol
3. Wash cells with PBS followed by trypsin (000.5%) for 1 min to remove excess PFF bound on the extracellular surface.

SECTION: αS PFF Clearance Assay Protocol
4. Incubate with fresh media in presence or absence of other drugs/inhibitor treatment for desired duration prior to lysate collection.
TITLE: Expansion of mouse embryonic fibroblasts (MEFs) for hPSC cultures
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the expansion of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture.

General notes
1. Throughout this protocol, 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. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)
 






[BEFORE_START]
All cell culture plates which are used as feeders to maintain hPSCs are coated for at least 1 hour with autoclaved 0.2% gelatin solution at room temperature. Remove gelatin solution immediately before plating MEF cells. 

0.2% Gelatin Solution
 

 
 

[STEPS]
1. Wash the plates twice with DPBS 
2. Add Trypsin and incubate for 5 min (37°C; 5% CO2)

3. Add MEF medium to neutralize the Trypsin and collect the solution into a conical tube.
4. Centrifuge at 250 x g, 5 min 

5. Remove the supernatant. Re-suspend the cell pellet in fresh MEF medium to plate on gelatin-coated plates (Dilution ratio 1:4) and maintain in a humidified incubator (37°C; 5% CO2). 
6. MEFs need to be passaged once confluent (every 3-4 days) and can be expanded up to passage 4 (P4) and frozen before or after inactivation using irradiation or Mitomycin C treatment. 

For more information on freezing MEFs, as well as irradiation and Mitomycin C treatment of MEFs, refer to the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture." A link to this collection can be found in the title section of this protocol, located above
3.1. MEF medium
TITLE:          ELISA for quantification of CXC motif chemokine ligand 2 (CXCL2) in human serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human CXCL2 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human CXCL2 present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-CXCL2 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-CXCL2 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of CXCL2 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human CXCL2 standard dilutions and the human CXCL2 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: CTAB DNA Extraction for high quality/molecular weight DNA
AUTHORS: Yaowu Yuan

[STEPS]
?. Grind plant tissue in a mortar cooled with liquid nitrogen.
?. Add .
[CTAB DNA Extraction buffer]
?. Wait until it warms up and becomes a green paste, then transfer to an eppie tube.
?. Incubate the CTAB/plant extract mixture for  at  in the heat block and invert to mix throughout the 15 minutes.
?. Add : IsoAmyl Alcohol (24:1) in the hood and mix the solution by inverting the tubes (do not vortex).
[Chloroform]
?. Centrifuge at   for .
?. Transfer the upper aqueous phase only to a new eppie tube (~).
500 µl
?. Add RNase A (10 µg/ml).
of 1mg/ml stock if you have   of sample.
?. Incubate at  for .
?. Add  followed by  and invert to mix.
[7.5M Ammonium acetate]
[ice cold 100% ethanol ]
?. Put tubes in  for 1 hour (or longer) to precipitate the DNA.
[freezer]
?. Centrifuge at  for .
You should see a pellet at the bottom (align the tubes so that you know where the pellet is in case you can’t see it very well).
?. Remove the supernatant and wash the DNA pellet as follows. (1/2)
?. Add  . (1/2)
[ice cold 70% ethanol]
?. Centrifuge at  for . (1/2)
?. Remove the supernatant and wash the DNA pellet as follows. (2/2)
?. Add  . (2/2)
[ice cold 70% ethanol]
?. Centrifuge at  for . (2/2)
?. Remove all the supernatant and allow the DNA pellet to dry in the hood (approx. ).
Do not over dry the pellet since it will be hard to re-dissolve.
?. Resuspend the DNA in .
[dH2O]
?. NanoDrop the sample to estimate the concentration.
Alternatively the DNA can be run on a gel to estimate the concentration or size of the DNA. 
Running a 0.4% gel overnight with the lambda DNA mono-cut ladder can give you an estimate of the size (it still doesn’t separate the large bands very well).
TITLE: Snap-Frozen Tissue Preparation
AUTHORS: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill

[DESCRIPTION]
This protocol describes preservation of tissue by snap-freezing.  Biospecimens preserved with this protocol will be suitable for downstream analysis of DNA, RNA, protein, and morphology endpoints.  Multiple workflows can be used for snap freezing including using placing tissue in a vessel either with or without media and exposing it to liquid nitrogen vapor, immersing it in liquid nitrogen, or placing it on  dry ice with a cooling device (Micke et al., 2006).  The workflow pursued depends on the desired balance between feasibility, time, and cost as well as the possible downstream analyses performed.  The snap-freezing protocol detailed below may not be ideal for downstream assays that require intact tissue morphology (Engel, Vaught, & Moore, 2014) (see NCI Biospecimen Evidence-Based Practices).

Micke, P., Ohshima, M., Tahmasebpoor, S., Ren, Z.-P., Ostman, A., Pontén, F., & Botling, J. (2006). Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens.Laboratory Investigation; a Journal of Technical Methods and Pathology,86(2), 202–211. https://doi.org/10.1038/labinvest.3700372

Engel, K. B., Vaught, J., & Moore, H. M. (2014). National Cancer Institute Biospecimen Evidence-Based Practices: A novel approach to pre-analytical standardization.Biopreservation and Biobanking,12(2), 148–150. https://doi.org/10.1089/bio.2013.0091

[STEPS]
1. Place pre-weighed piece of tissue in either a 1.5mL Eppendorf tube or 1.5mL cryo tube.
2. Quick freeze by immediately placing tube on dry ice.
3. Transfer tube to a -80℃ ultra-low freezer for Biobanking or until ready for downstream processing.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for the detection of phosphorylated antigens via flow cytometry without the use of commercially available kits. It is adapted from the publication linked after the description</p>

[BEFORE_START]
<p>Prepare IMDM (complete):</p>
<p> </p>
<p>Add to the 500 ml IMDM Bottle:</p>
<p>25 ml fetal bovine serum (final concentration: 5%)</p>
<p>5 ml L-Glutamine (final concentration 2 mM)</p>
<p>500 µl Gentamicin (final concentration 50 µg/ml)</p>
<p>500 µl 2-Mercaptoethanol (final concentraiton 50 µM)</p>
<p> </p>
<p>Prepare PBS with 5% BSA. Dilute part of it to 0,5%.</p>
<p> </p>
<p>Prepare 1mM Ionomycin stocks in DMSO.</p>
<p>Prepare 10 µg/ml PMA Stocks in DMSO.</p>
<p>Prepare 8% PFA Solution in 1xPBS.</p>
<p>Cool methanol to 4 °C. </p>
<p>Prepare ice bath under the fume hood for incubation with methanol.</p>
<p> </p>
<p>Cool down the centrifuge.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Endothelin is the most powerful vasoconstrictor. It exists not only in the vascular endothelium, but also in various tissues and cells. It is an important factor in regulating cardiovascular function and maintains basal vascular tone and heart, and plays an important role in the vascular system homeostasis. The endothelin system includes three peptide ligands (<a href="https://www.creativebiomart.net/symbolsearch_EDN1.htm" target="_blank" rel="noopener noreferrer">EDN1</a>, EDN2, and <a href="https://www.creativebiomart.net/symbolsearch_EDN3.htm" target="_blank" rel="noopener noreferrer">EDN3</a>) and two G protein-coupled receptors (EDTRA, <a href="https://www.creativebiomart.net/symbolsearch_EDNRB.htm" target="_blank" rel="noopener noreferrer">EDTRB</a>).</p>

[STEPS]
?.
TITLE: SPARC Cat - Sham Control Chronic Cat 3, Day 0
AUTHORS: Brett       Hanzlicek, Margot Damaser

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a procedure for a sham control chronic cat experiment (Day 0) 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 basic surgery, urodynamics and imaging for Day 0 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.
?. [Cystogram - DYNA CT]
Use 1:5 dilution of contrast to saline to visualize bladder.Take a 3D CT image with 30ml contrast:saline in the bladder
?. [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  LabView
?. [Urodynamics/Cystometry 1]
Empty BladderCat transitioned back to isoflurane
?. [Surgery]
See protocol titled "SPARC Cat surgery Day 0" for details on accessing and opening the bladder for device insertion.
?. [Surgery]
Place device in bladder,  suture the bladder closed with 4-5 stitches and add 25 ml of dilute contrast through the catheter.  Check for leaks and place bladder back into body.
?. [Surgery]
Take a picture with DYNA-CT and a 3D CT
?. [Surgery]
Remove stitches from bladder, remove device and suture the bladder and abdomen per protocol titled "SPARC Cat surgery Day 0"
?. [Urodynamics/cystometry 2]
Fill bladder with saline at 2ml/min
?. [Urodynamics/cystometry 2]
Fill bladder with saline and record pressure using pressure transducer connected to  LabView
?. [Wake and return cat]
Wait for cat to wake up and return to housing site.
TITLE: Human Pregnant Uterine Myometrium Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC
AUTHORS: Valentina  Stanley, Scott   Lindsay-Hewett, Mana   Parast, Louise  Laurent

[DESCRIPTION]
Human pregnant uterine myometrium tissue collection and storage protocol for HuBMAP's UCSD Female Reproductive TMC.

[STEPS]
SECTION: Preparation
1. At the time of c-section, obtain a full-thickness uterine myometrial biopsy from the superior edge of the incision site, prior to closure (about 2.0cm x 1.0cmx 0.5cm). Collect the tissue into an empty sterile cup.
SECTION: Preparation
2. Wash the tissue in cold PBS.
SECTION: Preparation
3. Place tissue on the guaze pad keeping the serosal (smooth) side pointing up and endomyometrial side pointing down. Take a photo. Split the tissue in half horizontally, dividing the serosal side from the endomyometrial side.
   

SECTION: Preparation
4. Cut each side into smaller chunks, maintaining the medial to lateral and serosal to endomyometrial orientation.
  

SECTION: Preserving the serosal side
5. Collect cross sections of the serosal side into 10% formalin (x1), RNAlater (x3) and to be snap frozen (x3). The pieces should be about 5mm each, depending on the total size of the tissue. If there is an excess of tissue, collect more of each type. S1 should be most medial, then S2, then S3 should be most lateral.
SECTION: Preserving the endomyometrial side
6. Collect cross sections of the endomyometrial side into 10% formalin (x1), RNAlater (x3) and to be snap frozen (x3). The pieces should be about 5mm each, depending on the total size of the tissue. If there is an excess of tissue, collect more of each type. E1 should be most medial, then E2, then E3 should be most lateral. 
SECTION: Flash-freezing
7. Drop the snap frozen labeled tubes into liquid Nitrogen. Leave for ~2-10 minutes, then store in -80C freezer.
SECTION: Processing with RNAlater
8. Place RNAlater tubes into a 4C fridge. Let these sit for 24-48 hours, then remove the RNAlater with a sterile transfer pipette and store in a -80C freezer.
SECTION: Formalin fixing
9. After one week in formalin, place each formalin-fixed piece into a labeled cassette to be processed into FFPE blocks.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The growing of microbrewerys in Brazil has imposing an increase in the production of different supplies for brewing, iincluding yeast biomass for beer fermentation. In Brazil,only four companies produce and sell yeasts for microbreweries, and only two companies are able to attend microbreweries. Additionaly, these companies should be able to deliver yeast cells to different states and cities in Brazil. During transport, the yeast cells are exposed to thermical stress (due to the lack of temperature control) and chronological aging (due to the long period of storage and transport). When delivered to the microbreweries, those yeast cells display problems during beer fermentation as the consequence of a loweryeast vitality and viability and affecting beer’s quality. Thus, in this work a new colorimetric assay was developed to access the vitality of yeast cells by using resazurin. Resazurin is a highly dichromatic dye, showing a colorimetric change proportional to the yeast cellvitality that can be easily visualized by naked eye. To evalute the potential of resazurin to differentiate yeast cells with low and high vitality, different yeast ale and lager strains (US-05 and W34/70, respectively) were submitted to heat shock stress and their vitality was accessed by resazurin, testing different variables as concentration of work solution, cellular density and incubation time. The resazurin data, supported by acidification power test, showed a vitality decrese in cells submited to heat shock stress. Futhermore, the sensibility of the resazurin assay was explored during chronological aging of yeast cells. The assay was capable to detect differences of vitality and viability between yeast strains. In addition, the potencial of the resazurin test to predict the fermentative capacity of stressed and non-estressed yeast cells was observed in wort attenuation assays, comfirming the aplicability of resazurin in the microbrewery context.</p>

[STEPS]