eamag/protocols-with-steps huggingface dataset:
TITLE: Algal DNA extraction for HMW Nanopore sequencing
AUTHORS: Robert Auber, Jennifer Wisecaver

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol adapted for a haptophte algae. It is a single cellular organism lacking any type of silica or calcium carbonate armor. If your organism doesn't lyse by a simple snap freeze, some type of grinding step is recommended. Confirmation of properly extracting the nuclei can be done with propidium iodide staining and an epiflourescent microscope. The actual DNA extraction gives ultra long reads (greater than 200kb) by using a circulomics NanoBind kit.</div></div>

[STEPS]
?. [Prepare equipment and reagents]
Set water bath to 74°C
?. [Prepare equipment and reagents]
Chill centrifuge to 4°C
?. [Prepare equipment and reagents]
Add BME to premade NIB (Per Sample 35 μl BME into 35 ml premade NIB + Aliquot  1.8 ml NIB and add 200 μl Triton X-100)
?. [Prepare equipment and reagents]
Cool NIB to 0°C
?. [Prepare equipment and reagents]
Cool NIB + Triton X to 0°C
?. [Prepare equipment and reagents]
Get liquid nitrogen
?. [Prepare equipment and reagents]
Chill four 50 ml conical tubes (per sample) on ice
?. [Cell Lysis]
?. [Cell Lysis]
Transfer 20-50 ml of culture to a pre-chilled 50 ml conical tube and centrifuge at 4°C at 2000g for 10 min to pellet cells.
?. [Cell Lysis]
Discard the supernatant and snap freeze the conical tube in liquid nitrogen.
?. [Cell Lysis]
Place cells on ice and add 10 ml of ice cold NIB.
?. [Cell Lysis]
Mix by slowly pipetting up and down with a 5 ml pipettor until the mixture is smooth and not clumpy
?. [Filter to remove particulate material]
Place a 100 μm filter on top of a pre-chilled 50 ml conical tube
?. [Filter to remove particulate material]
Slowly run the sample through the filter into the clean tube
?. [Filter to remove particulate material]
Wash the filter with 10 ml of ice cold NIB
?. [Filter to remove particulate material]
Place a 70 μm filter on top of a second pre-chilled 50 ml conical tube
?. [Filter to remove particulate material]
Slowly run the sample through the filter into the clean tube
?. [Filter to remove particulate material]
Wash the filter with 10 ml of ice cold NIB
?. [Filter to remove particulate material]
Place a 40 μm filter on top of a third pre-chilled 50 ml conical tube
?. [Filter to remove particulate material]
Slowly run the sample through the filter into the clean tube (do not wash with fresh NIB)
?. [Filter to remove particulate material]
Add 1.5 ml of ice cold NIB+TritonX
?. [Filter to remove particulate material]
Centrifuge at 4°C at 500g for 10 min to pellet nuclei.
?. [Filter to remove particulate material]
Discard the supernatant
?. [Extract DNA from Nuclei]
D. Extract DNA by referring to Circulomics Plant Nuclei Big DNA Kit protocolhttps://www.circulomics.com/store/Nanobind-Plant-Nuclei-Big-DNA-Kit-Alpha-Version-p99924200
TITLE: Th17 Polarization of Mouse CD4+ Cells
AUTHORS: Sam Li

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

[STEPS]
?. [Isolation of CD4+ Cells From Lymph Nodes]
Harvest lymph nodes (superficial cervical, mandibular, axillary, inguinal, and mesenteric) from mice.
?. [Isolation of CD4+ Cells From Lymph Nodes]
Tease lymph nodes through a sterile 70-µm nylon cell strainer to obtain single-cell suspensions in complete IMDM containing 10% FCS (complete medium).
?. [Isolation of CD4+ Cells From Lymph Nodes]
Resuspend cells in complete medium and use your favorite method to isolate CD4+cells. Consider using our MojoSort™ Mouse CD4 T Cell Isolation Kit.
?. [Th17 Polarization of CD4+ Cells]
On day 0, coat 60 x 15mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11 (5µg/ml). Incubate at 37°C for 2 hours or 4°C overnight. Aseptically decant antibody solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
?. [Th17 Polarization of CD4+ Cells]
Plate CD4+cells at 10 x 106/5 ml/dish. Culture cells for 4 days in the presence of anti-mouse CD28, clone 37.51 (5µg/mL), recombinant mouse IL-6 (50ng/mL), recombinant human TGF-β1 (1ng/mL), recombinant mouse IL-23 (5ng/ml), anti-mouse IL-4 (10µg/mL), and anti-mouse IFN-γ (10µg/mL).
?. [Th17 Polarization of CD4+ Cells]
On day 3, slowly add 5ml of fresh media along with same the concentration of antibodies/cytokines as used on day 0.
?. [Th17 Polarization of CD4+ Cells]
On day 4, wash cells once and then restimulate in complete medium with 500ng/ml PMA and 500ng/mL ionomycin, in the presence of Brefeldin A (If you are looking for IL-21 production, use monensin) for 4-5 hours.
?. [Th17 Polarization of CD4+ Cells]
After harvesting, the cells are ready for staining.Tip: Recombinant human TGF-β is effective for stimulating mouse cells.
TITLE: Gentle cell extraction from rocks for downstream single cell sorting and sequencing
AUTHORS: Jackie Goordial, Beth Orcutt, Tim Dangelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Method for extracting cells in a gentle manner from rocks for downstream single cell sorting and sequencing. Starting material for the protocol is rock in a 15 mL centrifuge tube, stored frozen in 5 mL of Glycerol TE. GlyTE stock recipe is provided in materials section. </div></div>

[STEPS]
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube]
Add EDTA to 15 mL tube ( containing 5 mL glyTE and 5 g crushed rock), add EDTA for final concentration of 10 mM.
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube]
Vortex two tubes at time horizontally in tube holder of the Vortex-Genie-2. Vortex for 5 min at speed 7 (too high of a speed could allow the rock particles to cause damage to the cells)
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube]
Sonicate, two tubes at a time in ice water, for 1 Minute
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube]
Centrifuge at 1,000 RPM for 5 minutes to pellet rock particles. Transfer the supernatant to a fresh 15 ml Falcon tube
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube]
Centrifuge the supernatant 2ml at time @ 10,000 RPM for 5 minutes in 2 x 2 ml centrifuge tubes. One tube for sorting, and another as replicate or microscopy. Approximately 2.5 ml will be pelleted in each tube over two rounds of centrifugation (2 ml, 2ml, ~ 250 ul). Remove supernatant each time, with caution, using a pipette. Do Not Pour Off Supernatant, cellular material is not always well pelleted and can be lost easily.
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube]
a. For one tube (for later sorting) Resuspend the pellet in 1ml of 1X GlyTE. Can dilute with 1X PBS. Store in freezer.  b. For second tube (for microscopy) – stain with SYBR Green, and filter on 0.2 um polycarbonate filter, rinse and dry filter, then mount on glass slide with Citifluor antifade mounting solution. Count cells, or store slide at 4C until ready to count cells
TITLE: RNA collection, cDNA conversion and qPCR (SH-SY5Y cells)
AUTHORS: Stephanie Vrijsen, Peter Vangheluwe

[DESCRIPTION]
This protocol describes the isolation of RNA from SH-SY5Y cells and the subsequent conversion to cDNA for qPCR. 

[STEPS]
SECTION: RNA collection
1. Cells were seeded in 10 cm dishes and used for collection when reaching 70-80% confluency. 
(e.g. 3 million cells for collection after 2880 min) 

SECTION: cDNA conversion
11. Convert RNA to cDNA using the High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific). 
SECTION: RNA collection
4. Scrape and collect cells in PBS(-/-). 
SECTION: RNA collection
5. Spin down cells (450xg, 5 min). 

SECTION: RNA collection
6. Wash with PBS(-/-). 
SECTION: RNA collection
2. Remove medium. 
SECTION: RNA collection
3. Wash once with PBS (-/-). 
SECTION: RNA collection
7. Spin down cells (450xg, 5 min). 
SECTION: RNA collection
8. Remove supernatant. 
SECTION: RNA collection
9. Isolate RNA following the instructions of the NucleoSpin RNA Plus kit (740984.50, Macherey-Nagel). 
! Use a separate, desinfected area to isolate RNA. Use filter tips and dedicated pipets for RNA work. 
SECTION: RNA collection
10. Determine the concentration and the purity of the isolated RNA using a Nanodrop spectrometer. 
SECTION: cDNA conversion
11.2. Prepare a 2x Mastermix: 
      
  Volume
  (µl)
  Component
  
  2
  µl RT buffer
  
  0,8
  µl dNTPs (100 µM)
  
  2
  µl random primers
  (10x)
  
  1
  µl Multiscribe
  transcriptase (10u/µl)
  
  4,2
  µl AD
  
 
 

 
SECTION: cDNA conversion
11.1. Prepare 5 µg RNA in 20 µl total volume (dilute with RNase free water). 
SECTION: cDNA conversion
11.3. Add 10 µl of the mastermix to 10 µl of the RNA dilution. 
SECTION: cDNA conversion
11.4. Perform a quick vortex and spin down using a table-top centrifuge. 
SECTION: cDNA conversion
11.5. Start program for RNA to cDNA conversion: 
  
 
          
  
  Step 1
  Step 2
  Step 3
  Step 4
  
  Temp (°C)
  25
  37
  85
  4
  
  Time
  10 min
  120 min
  5 sec
  ∞
  
 




 
14. Prepare a ten-fold dilution of the cDNA samples in duplicates (5 µl cDNA per well). 
Include a negative control where the cDNA is exchanged by an equivalent volume of water. 
SECTION: qPCR
13. Prepare a master mix containing per sample: 
10 µl SYBR Green master mix (Roche)
1 µl of 5 µM forward primer
1 µl of 5 µM reverse primer
3 µl water
15. Add 15 µl of the master mix to each well. 
SECTION: qPCR
12. Prepare a serial dilution of the sample that you choose as standard (1/5, 1/25, 1/125, 1/625, 1/3125). Include water as a negative control. 
Pipet in duplo in a 96-well plate (5 µl per well). 
17. Start the qPCR reaction:
95 °C for 10 min
50 cycles at 95 °C for 10 s 
55 °C for 30 s 
95 °C for 1 min 

Determine a melting curve from 55 to 95 °C. 
16. Cover plate with a film and spin samples down.
TITLE: Concentration technique for Viable but Non-Culturable organisms 
AUTHORS: Muhammad Muhsin Fathuddin, Solomon John Obidah

[DESCRIPTION]
This protocol is based on existing protocols, such as those for resuscitating microbes, freeze-drying, and selective isolation. It merges the parts of the three protocols to create a new protocol for isolating microbes via resuscitation, centrifugation, and selective isolation. The first part involves resuscitating the microbes; the second part subjects the medium to centrifugation (concentration of the microbes); the third and final part involves the selective growth in a selective broth and subsequent growth on selective agar.

[BEFORE_START]
Wear necessary safety gear before commencing the work

[GUIDELINES]
Laboratory protocols must be followed, and the environment must be kept as sterile as possible.

[STEPS]
SECTION: Resuscitation of Microorganism
1. To the resuscitation broth media (e.g., peptone water, tryptic soy broth, Luria-Bertani broth, etc.), add the sample, making a 10% solution, i.e.,10 mg in90 mL.

SECTION: Resuscitation of Microorganism
2. Incubate the broth culture at 37oC for 24-48 hours
SECTION: Selective medium growth of desired organism
5. Mix cell pellets with the desired selective medium broth. Suspend cells thoroughly by using a vortex mix  until complete cell resuspension is achieved aseptically.

SECTION: Selective medium growth of desired organism
6. Then, incubate at the desired temperature1440 min, which can be extended up to10080 min.
SECTION: Selective medium growth of desired organism
7. From the above selective broth, subculture onto a selective agar for the desired organism.
SECTION: Selective medium growth of desired organism
8. incubate the selective agar plate at the required temperature for 24–48 hours. then observe for the desired growth and colony morphologies
SECTION: Centrifugation of the resuscitation media
3. The grown broth culture is then centrifuged5000 rpm, 30 min, 4 °C
SECTION: Centrifugation of the resuscitation media
4. Then, concentrate the cell suspension and decant the culture supernatant.
SECTION: Selective medium growth of desired organism
9. Store the isolate for further tests (Biochemical, Molecular, etc.)
TITLE: Nuclei Isolation
AUTHORS: amanda schneeweis

[DESCRIPTION]
nuclei isolation for snRNA-seq

[STEPS]
1. Prepare buffers and filter sterilize, add RNAse inhibitor fresh NP40 Lysis Buffer (NST): 0.1% NP-40 Alternative (or NP-40), 10 millimolar (mM) Tris, 146 millimolar (mM) NaCl, 1 millimolar (mM)CaCl2, 21mM MgCl2, 40U/mL of Protector RNAse inhibitor (add fresh day of) ST Wash Buffer: (10mM Tris, 146 Mass Percent NaCl, 1 millimolar (mM) CaCl2, 21mM MgCl2), 0.01% BSA, 40U/mL of Protector RNAse inhibitor (add fresh day of) ST Staining buffer (ST-SB): 2%BSA, 0.02%Tween-20, 10 millimolar (mM) Tris, 146 millimolar (mM)NaCl, 1mM CaCl2, 21mM MgCl2), 40U/mL of Protector RNAse inhibitor (add fresh day of) Note: Keep tissues/homogenate and buffers on ice throughout the protocol. Pre-cool the centrifuge to 4 °Cand keep at 4 °C for all steps.

2. Tissue collection a) Sacrifice and rapidly decapitate mice. Using chilled brain matrix (Ted Pella), cut a thick coronal section from 5mm back from start of cortex until start of cerebellum. Store section in ice-cold Hibernate-A media in 10cm plate on ice until all brains have been dissected. b) Use a razor blade to dissect out midbrain and collect tissue, placing directly into dounce homogenizer.
3. Tissue lysis and homogenizing a) For each sample to barcode and pool: prepare a separate homogenizer and douncing pestles (loose and tight). Add 1 mL NST buffer to the tube with tissue and transfer to dounce homogenizer and keep on ice. b) with a total volume of 1 mL, dounce 20 times with the loose pestle followed by 20 times with the tight pestle. c) Add 1 mL of ST wash buffer, filter through 30µm filters (Milentyi Biotec 130-041-407) and transfer filtered homogenate to a 15mL tube. d) Rinse the homogenizer with 3x 1 mLof ST wash buffer, filter through 30µm filters and add to the filtered homogenate to add up to a final volume of 5 mL. e) Immediately spin down at 500 g for 5 min at 4 °C to pellet the nuclei in swing bucket rotor f) Remove supernatant g) Resuspend nuclei in 200µl of ST-SB and transfer to lo-bind 1.5 mLtube and wash out the original tube with an additional 1 mL and transfer to the same 1.5ml tube. h) Wash by spinning down for 5 min at 500 g at 4 °Cand resuspending in 1.2 mL ST-SB twice, for a total of 3 washes i) resuspend in 1 mL ST-SB

4. Proceed with sorting nuclei for GFP+ nuclei via MACS Tyto
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol for DNA Assembly using the&nbsp;NEBuilder&reg; HiFi DNA Assembly Master Mix (E2621).

[GUIDELINES]
<strong>Optimal Quantities</strong><br /><br />NEB recommends a total of 0.03&ndash;0.2 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector, and 0.2&ndash;0.5 pmols of DNA fragments when 4&ndash;6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend the following formula, or using the tool,<a href="https://www.neb.com/external-links/nebiocalculator" target="_blank" data-ga-category="External Link" data-ga-action="https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol" data-ga-label="https://www.neb.com/external-links/nebiocalculator">NEBiocalculator</a>.<br /><br />pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)<br />50 ng of 5000 bp dsDNA is about 0.015 pmols<br />50 ng of 500 bp dsDNA is about 0.15 pmols<br /><br />The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm or estimated from agarose gel electrophoresis followed by ethidium bromide staining.

[STEPS]
?. 
?. 
?. 
?.
TITLE: Tutorial to use the Galaxy/coral SNP STAGdb  workflow
AUTHORS: Iliana Baums, Sheila Kitchen, Greg vonKustner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Tutorial to use the Galaxy coral SNP STAGdb workflow </span><a href="https://coralsnp.science.psu.edu/galaxy" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://coralsnp.science.psu.edu/galaxy</span></a></div></div>

[STEPS]
TITLE: Cryo-EM sample preparation for PI3KC3-C1~RAB1A complex
AUTHORS: Annan SI Cook

[DESCRIPTION]
This protocol details cryo-EM sample preparation for PI3KC3-C1~RAB1A complex.

[STEPS]
SECTION: GTP Loading of RAB1A
1. Strip the protein of its bound divalent cations by incubating RAB1A(Q70L) with 5 millimolar (mM) EDTA at Room temperature for 30 min.
SECTION: GTP Loading of RAB1A
2. Add a large molar excess (50 millimolar (mM)) of GTP to the protein.

SECTION: GTP Loading of RAB1A
3. Incubate the mixture for an additional 30 min to allow GTP loading.

SECTION: GTP Loading of RAB1A
4. Quench the reaction by adding 20 millimolar (mM) MgCl2 at Room temperature for 20 min.
SECTION: Desalting of GTP-loaded RAB1A
5. Purify the GTP-loaded RAB1A through a PD-10 column equilibrated with a final buffer containing 25 millimolar (mM) HEPES pH 7.5, 150 millimolar (mM) NaCl, 2 millimolar (mM) MgCl2, and 25 millimolar (mM) TCEP.
SECTION: Preparation of PI3KC3-C1~RAB1A Complex
6. Prepare a 1:20 molar ratio of PI3KC3-C1 to RAB1A (0.55 micromolar (µM) PI3KC3-C1: 11 micromolar (µM) RAB1A).
SECTION: Preparation of PI3KC3-C1~RAB1A Complex
7. Incubate the mixture for 60 min at Room temperature to allow complex formation.
TITLE: IV catheter making
AUTHORS: McKenzie Pavlich, Olivier George

[STEPS]
?. 
?. Bend cannulas into a curved 90° angle making sure that the curve is not too sharp (1.5 cm from tip of bending tool)Create a ruler from tape and mark all important sections Start >> 15 cm >> Stop        ** SMALL tubing should be a total of 15 cm  Make mark at 4 cm in from stopping point      ** Silicone ball will be placed here Make a mark 2 cm from starting point     ** LARGE tubing should be a total of 2 cm
?. 
?. Cut and mark tubes   * Small tubing 15 cm* Large tubing 2 cm* Mark for silicon ball at 4cm from ending point of small tubingWhen finished cutting tubing, dip starting end of the small tubing (make sure not ending side) into CitriSolv to temporarily increase the diameter of the tube. Also fully submerge larger tubing in the CitriSolv. Let sit for 10 minutesThread small tubing over the bent end of the cannula and make sure that the tubing is touching the start of threaded portion of the cannula. (It will shrink as it dries and this will ensure it is as close as possible to the cannula when dried)Let small tubing dry for around 2 hoursThread larger tubing on the smaller tubing and make sure it is touching the start of the threaded portion of the cannula as wellLet dry over night
?. 
?. Place the cannulas in the mold. Have the threads of the cannula sitting into the threads of the mold
?. 
?. Place top of mold onto the other side of the mold that is holding the cannulas. Make sure the seal of the mold is tight, has no gaps and doesn’t wobblePut whole mold into the vice. Make sure the vice is level and not tiltedAfter putting the mold into the vice, bring the following supplies to the hood:Vice with moldSpatulaDental cement powder & liquidMesh piecesWeigh boats for mixing the dental cementSiliconMix dental cement powder with the liquid to create the dental cement
?. 
?. Quickly fill each well with the mixture using the spatula. Make sure that the mixture has sunk to the bottom of the mold before adding more
?. 
?. Place mesh on top of wet cement then add a bit more to seal mesh in place. Let dry at least 30 minutes and then take them out of mold
?. Cut mesh into round shape
?. Shave off any rough edges around the base/port of the catheter, then flush them with water to ensure no leaks or holes
TITLE: 10Xv3.1 Genomics Sample Processing
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used for the rapid generation of 3’ transcriptomic-NGS-ready- single-cell-libraries from pools of cells.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number's 1U01MH114812-01 and 1U19MH114830-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: UC Davis - Glucose Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Glucose is oxidized by glucose oxidase to gluconic acid and hydrogen peroxide. The hydrogen peroxide reacts in the presence of peroxidase with HBA and 4-aminoantipyrine forming a red quinoneimine dye. The intensity of the color formed is proportional to the glucose concentration and can be measured photometrically between 460 and 560 nm.</div></div>

[STEPS]
?. Reconstitute powdered reagent with only 25 ml of distilled water to make a 2X solution.
?. Add 3 μl of calibrator and sample to each well.
?. Add 150 μl of PBS to each well. Read at 540 nm.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Add 150 μl of 2X reagent to each well. Incubate at 37°C for 10 minutes. Read at 540 nm.
?. Subtract blank readings from final readings. The assay will be linear so the unknown samples can be calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.
TITLE: Echocardiography: Mouse
AUTHORS: E. Dale Abel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">This protocol describes the procedure used by the DiaComp for transthoracic echocardiography in awake mice. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bidt4kcn.jpg" /></div></div>

[STEPS]
?. We perform transthoracic echocardiography in awake mice. If anesthesia is required we currently favor the use of inhaled isoflurane (1%), delivered via nose cone. The anesthesia flow can be titrated to minimize any reduction in heart rate. If inhaled anesthesia is not available, we have also had success with avertin (tribromoethanol)(0.2 ml/10 g body weight) or chloral hydrate (0.5 mg/g body weight). Heart rates are generally maintained at more than 400 per minute with these regimens. The chest hair is removed with a topical depilatory agent. Limb leads were attached for electrocardiogram gating, and the animals are imaged in the left lateral decubitus position with a 13-MHz linear probe (Vivid FiVe; GE Medical Systems, Milwaukee, Wisconsin, USA). Two-dimensional images are recorded in parasternal long- and short-axis projections with guided M-mode recordings at the midventricular level in both views. Left ventricular (LV) cavity size and wall thickness are measured in at least three beats from each projection and averaged. LV wall thickness [interventricular septum (IVS) and posterior wall (PW) thickness] and internal dimensions at diastole and systole (LVIDd and LVIDs, respectively) are measured. LV fractional shortening [(LVIDd – LVIDs)/LVIDd], relative wall thickness [(IVS thickness + PW thickness)/LVIDd], and LV mass [1.05 (IVS thickness + LVIDd + PW thickness)³  – LVIDd³ ] are calculated from the M-mode measurements.