eamag/protocols-with-steps huggingface dataset:
TITLE: Sample preparation to mass spectrometer using ZipTipC18
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ZipTip is a 10 µL pipette tip that aims to concentrate and purify from femtomols to picomols protein and oligopeptide .</div></div>

[STEPS]
?. [Prepae the materials and workspace]
Calibrate the P-10 pipette to 10 μL.
?. [Prepae the materials and workspace]
Name 4 microtubes of 1.5 mL with the respective reagents:ACN100% (I)TFA0.1% (I)TFA 0.1% (II)ACN100% (II)Add 1 mL of the respective solutions to the tubes above.
?. [Prepae the materials and workspace]
Name new 0.2 mL microtubes with sample names followed by “zt”.
?. [Prepae the materials and workspace]
Aliquot 50 μL of ACN 100% in each “zt” tube.
?. [NOTE]
During the entire process, no air should enter through ZipTip. Pipetting should be done slowly to maximize chemical interactions
?. [Procedure]
Pipette the acetonitrile from the ACN100% (I) tube 10 times, discarding the contents on the kimwipes
?. [Prepae the materials and workspace]
Fit the ZipTip with the pipette already attached, so as not to pass air through the "chromatographic column". Hold down until you start the procedure with the first pipetting.
?. [Procedure]
Pipette the acetonitrile from the TFA0.1% (I) tube 10 times, discarding the contents on the kimwipes
?. [Procedure]
Homogenize the sample with ZipTip 20 times
?. [Procedure]
Pipette the acetonitrile from the TFA0.1% (II) tube 10 times, discarding the contents on the kimwipes
?. [Procedure]
Homogenize 20 times in the “zt” tube, already with 50 μL of ACN100%
?. [Procedure]
Shutdown: ACN100% (II)Pipette the acetonitrile from the ACN100% (II) 10 times, discarding in the “zt” tube.
TITLE: Preparation of human iPSC-derived cortical neuronal progenitors for transplantation into the rodent brain
AUTHORS: louise.cottle, Clare Parish

[DESCRIPTION]
This protocol describes how we prepare human iPSC-derived cells, that have been differentiated into cortical neuronal
progenitors, for transplantation into the brain of immunocompromised athymic mice. Neuronal progenitors mature within the mouse brain and are used to study the pathogenesis of Parkinson’s disease. 




[STEPS]
SECTION: Preparation of cell suspension
1. Prepare a Falcon tube with 2 mLbase cortical media plus Rock inhibitor (Ri, 1:1000
dilution)
Wash cells with 300 µL PBS -/- (gently run PBS down the side of each well)
Incubate cells in 300 µL Accutase (per well) at 37 °C to lift cells off wells in small
clumps
         - Monitor the Accutase incubation after 10 min; tap the plate to dislodge cells and look for cells lifting in a sheet before proceeding
        - Triturate 5 times to break the cells into clumps
        - Incubate clumps for a further 5 min at 37 °C
        - Triturate 10 times to break the cells into single cells and small clumps 

4. Transfer cells from the wells into the 15ml Falcon tube containing media + Ri (Step 1)
5. Rinse with plate with a further 300 µL of Accutase to ensure all cells are collected
6. Spin cells at 4 °C, 1300 rpm for 4 min
7. Discard supernatant
8. Resuspend cell pellet in 1-2ml base cortical media and add Ri (1:1000)
9. Count cells
       - Take 2 x 10 µLaliquot of cells in two separate tubes
Dilute cells 1:1 using 10 µL of trypan blue
       - Count cells in a 10 µL aliquot from each tube using a haemocytometer 
       - Calculate total number of cells (Total cells = Average count of quadrants x Dilution factor x Volume (ml) x 10^4)

10.  Calculate the total volume needed to resuspend cells to a final density of 100 000/uL
11.  Spin cells at 4 °C, 1300 rpm for 4 min
12.  Whilst cells are spinning, make up 1 mL media + Ri which will be used to resuspend the
cells for implantation surgery
13.  At end of the spin, collect tube and discard supernatant
14.  Using a P20, add 5 µL and resuspend pellet in base cortical media + Ri
15.  Transfer resuspended pellet to PCR tube and precisely measure the volume
16.  Add the remaining volume needed for cells to reach a density of 100 000 cells/ul as
calculated in step 10.
17.  Label the PCR tube containing the cells with details of the cell line and concentration
18.  Store cells on ice and transport to animal surgical room.

SECTION: Transplantation
2. Athymic mice undergo stereotactic surgery to receive a unilateral graft of 100,000
cortical progenitors in 1 µL volume. The surgical coordinates from Bregma are AP: +1, ML: +/-1.5, DV: -1.5

For full details of procedures for implantation of cell suspension can be found at https://www.protocols.io/view/transplantation-of-fetal-midbrain-dopamine-progeni-261ge4jq7v47/v1
TITLE: Live-cell imaging of the plasma membrane of Jurkat T cells
AUTHORS: Ezra Bruggeman, Markus Körbel

[DESCRIPTION]
This is a protocol for the preparation of a Jurkat T cells for live imaging of the plasma membrane. This protocol was used to generate the data shown in Figure 6 of the following publication:

Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479

[STEPS]
SECTION: Cell culture
1. J8 LFA-1 cells were incubated overnight (approximately 1080 min) in complete-RPMI (StableCell RPMI-1640 media, Sigma) supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % (v/v) HEPES buffer, and 1 % (v/v) pen/strep antibiotics.

SECTION: Cell culture
2. 1 mL of cells were collected by centrifugation and resuspended in phenol-red free RPMI supplemented with 1 % HEPES.

SECTION: Cell Labeling
3. Round coverslips were rinsed with IPA, MilliQ, dried, and Ar-plasma cleaned for 20 min.

SECTION: Cell Labeling
4. Grace Bio-Labs CultureWells were attached, and the slide was incubated with OKT3 antibody (provided by the Human Immunology Unit, WIMM, Oxford) for 30 min.

SECTION: Cell Labeling
5. The slide was washed 5 times with phenol-red free RPMI supplemented with 1 % HEPES.
SECTION: Cell Labeling
6. Perform a final wash with phenol-red free RPMI supplemented with 1 % HEPES and 200 nM NR4A (MemGlow‱ NR4A Membrane Polarity Probe, Cat. #MG06, Cytoskeleton Inc.) before imaging.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
It is important to plan the timing of the embryo collection in advance. To select a specific genotype, be sure to cross in the selectable balancer that will allow you to distinguish mutants from non-mutants. To prevent the collection of unfertilized animals into sample, it is helpful to select animals that have developed far enough through embryogenesis that auto-fluorescence of the gut can be observed.&nbsp;<br /><br />Example:To collect stage 17 Mcr[P1] animals: Setup a four hour collection and age for 17 hours at 25 degrees Celsius. For homozygous mutants, collect non-YFP animals and select only animals that have auto-fluorescence in the gut.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background: </strong>The spotted sea bass (<em>Lateolabrax maculatus</em>) is a valuable commercial fish that is widely cultured in China. While analyses using molecular markers and population genetics have been conducted, genomic resources are lacking.</p>
<p><strong>Finding: </strong>Here, we report a chromosome-scale assembly of the spotted sea bass genome by high-depth genome sequencing, assembly and annotation. The genome scale was 0.62 Gb with contig and scaffold N50 length to be 31 Kb and 1,040 Kb, respectively. Hi-C scaffolding of the genome resulted in 24 pseudochromosomes containing 77.68% of the total assembled sequences. A total of 132.38Mb repeat sequences were detected, accounting for 20.73% of the assemble genome. 22,015 protein-coding genes were predicted, of which 96.52% were homologous to proteins in various databases. In addition, we constructed a phylogenetic tree using 1,586 single-copy gene families and identified 125 unique gene families in the spotted sea bass genome.</p>
<p><strong>Conclusions: </strong>We assembled a spotted sea bass genome, which will be a valuable genomic resource to understand the biology of the spotted sea bass, and will also lead to the development of molecular breeding techniques to generate spotted sea bass with better economical traits.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for DNase I digestion of nuclei for 'double hit' DNase-SEQ analysis adapted from doi:10.1038/nmeth.2762</p>
<p> </p>
<p>As a general guide DNA should show moderate to light smearing after completing the protocol as seen the gel image in Figure 1.</p>
<p> </p>
<p><img src="https://s3.amazonaws.com/pr-journal/iuaeiee.png" width="160" height="160" data-src="https://s3.amazonaws.com/pr-journal/iuaeiee.png" data-ofn="DNase_digestion.png" /></p>
<p><strong>Figure 1:</strong> A representative 2% (w/v) LMP agarose gel of DNAse I treated nuclei (~2x10<sup>8</sup>) from <em>Sorghum bicolor.</em> To each sample the following amounts of DNase I were added: none, 7.5U, 12.5U.</p>
<p> </p>
<p>Over-digested samples will result in a high background signal from unspecific cutting of DNA and should be avoided. An example of an over-digested sample is provided in lane 5 of Figure 2.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/iubeiee.png" width="184" height="140" data-src="https://s3.amazonaws.com/pr-journal/iubeiee.png" data-ofn="DNase_digestion2.png" /></p>
<p><strong>Figure 2:</strong> A representative 2% (w/v) LMP agarose gel of DNAse I treated nuclei (~2x10<sup>8</sup>) from <em>Sorghum bicolor.</em> To each sample the following amounts of DNase I were added: none, 5,U 7.5U,10U, 12.5U.</p>
<p> </p>
<p>Gel fragments from ~50-600bp should be excised and DNA purified. From experience taking only smaller fragments (50-200 bp) caused difficulties in library preparation.</p>

[STEPS]
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TITLE: Anti oxidant and apoptotic activities of sitagliptin
AUTHORS: ruqaya alameen, ahsan f. bairam

[DESCRIPTION]


Abstract


Background: Hepatocellular carcinoma (HCC) is the most common and aggressive
type of liver cancer. Most chemotherapeutic medications nowadays imply
oxidative stress leading to toxicity, which raises the necessity to find agents
with better safety profiles against normal cells in addition to their
anticancer activity. Sitagliptin has been shown to possess antioxidant as well
as apoptotic properties by the specific suppression of dipeptidyl-peptidase 4,
a glycoprotein produced in many tissues that have been thought to promote tumorigenesis
and metastasis.


Methods: Five groups of cell-lines were
included: Control (untreated HepG2 cells); cisplatin treatment HepG2 cells;
sitagliptin treated HepG2 cells; combination of different concentrations of
cisplatin plus sitagliptin (250 μg/ml) treated HepG2 cells, and finally, combination
of different concentrations of sitagliptin plus cisplatin (25 μg/ml) treated
HepG2 cells. Then after incubation period for 48 hours, the supernatants were
collected to assess malondialdehyde (MDA) and B-cell
lymphoma-2 (BCL-2) by ELISA assay kits. Data were finally gathered and analyzed statistically.


Results: Our findings indicated that
sitagliptin decreased significantly the oxidative stress, particularly at high
concentrations, through decreasing the MDA level. In addition, sitagliptin
exhibited significant apoptotic activity against HepG2 cells through decreasing
BCL-2 level. In combination with cisplatin, sitagliptin significantly
potentiated the apoptotic effect and reduced the oxidative stress parameters.


Conclusion: Sitagliptin showed
apoptotic and antioxidant activity against HCC which may potentiate
chemotherapeutic agents like cisplatin, in addition to, reduce the oxidative
stress against normal cells.




[STEPS]
1. The cell were cultured into a 96-well plate with RPMI-1640 and then incubated for 24 hours 
2. The old medium was discarded and 200 μL of medium containing the test medicines was added 
3. Five primary groups were utilized including the control group, which were: control group, cisplatin-treated HepG2 group, sitagliptin-treated HepG2 group, group that received a different concentrations of cisplatin plus fixed concentration of sitagliptin (250 mcg) and group that received a different concentrations of sitagliptin plus fixed concentration of cisplatin (25 mcg)
4. the plates were incubated for 48 hours
5. the supernatant from each well was collected and frozen at -20 c
6. The MDA and BCL-2 levels were measured by specific ELISA assay kits
TITLE: DQ-BSA assay
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes the DQ BSA assay for measuring the lysosomal proteolytic activity.

[STEPS]
SECTION: DQ-BSA assay
1. Plate 100,000 mature macrophages or microglia or BMDM were seeded on Mattek glass bottom dishes.
SECTION: DQ-BSA assay
2. Next day treat cells with 50 nanomolar (nM) MLi2 or 250 nanomolar (nM) LRRK2-IN-1 for 120 min followed by 60 min treatment with 10 µg/mL DQ-BSA (Thermo Fisher Scientific, #D12051) and 50 µg/mL Alexa-488 BSA (Thermo Fisher Scientific #A13100) in the media containing inhibitors.

SECTION: DQ-BSA assay
3. After 3 hours wash the cells 3X with culture media.
SECTION: DQ-BSA assay
4. Then add 1 mL culture media to the washed cells and return to incubator for 10 min.

SECTION: DQ-BSA assay
5. Imaging was done on LSM 880.
TITLE: Peripheral Blood Mononuclear Cells Preparation
AUTHORS: John Davis Coakley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to prepare PBMCs for Stimulation experiment</div></div>

[STEPS]
?. Dilute whole blood 1:1 with 1% FCS HBSS Medium
?. Layer this diluted blood onto 10mls LymphoprepTM
?. Centrifuge for 25 minutes at 400G with acceleration and brake off
?. Discard the plasma and obtain the buffy coat
?. Top up buffy coat with 1% FCS, vortex and Centrifuge for 8 minutes at 1500RPM with brake on. Discard supernatant
?. Top up with 1% FCS, vortex and centrifuge for 8 minutes at 1500RPM with brake on. Discard supernatant.
?. Resuspend in 4-5mls of RPMI, and vortex.
?. Count live cells with haemocytometer slide after staining with ethidium bromide and acridine orange solution to detect dead and live cells, respectively
TITLE: Open field test
AUTHORS: Pranay Srivastava

[DESCRIPTION]
Open field test can be used to evaluate locomotor activity in rodents.

[STEPS]
1. Prepare the open field arena.
2. Allow the animal to acclimate to the testing environment for one hour.
3. The mice were placed in the plexiglass chamber (11 × 11 in with clear 8-in high walls) and were allowed to explore for a period of 10 min. 
4. The total distance travelled was measured with software Ethovision XT 9.0, Noldus Information Technology, The Netherlands.
TITLE: CHROMagar Malassezia Medium (CHROM)
AUTHORS: Amy Gladfelter

[DESCRIPTION]
Media recipe for growth of Malassezia species.

[STEPS]
1. CHROMagar Malassezia basal medium 56.3 g
 


2. Tween 40 10 mL

3. dH2O 1 L

4. Sterilize by autoclaving.
TITLE: Effect of antioxidants agents on Dentinal Tubular Penetration of EndoREZ sealer on sodium hypochlorite-treated root canal dentin: A Scanning electron microscopic study
AUTHORS: Sudhir  Varma

[DESCRIPTION]
According to ISO, the canals were enlarged to a size 30, 6% taper with ProTaper gold (Dentsply Sirona, Ballaigues, Switzerland) by crown down technique (Singh et al., 2015).Irrigation was performed with Saline and 17% EDTA in Group I. After biomechanical preparation, groups II, III, IV, and V were irrigated with 5 mL-Sodium hypochlorite (5.25%) and 5 mL-EDTA (17%) for 1 minute.
Irrigation was done on Groups III, IV, and V for an additional 10 minutes with 10% Ellagic acid, 5% Lycopene extract, and 5% Grape Seed Extract, respectively. After irrigation, the root canals were dried. The EndoREZ (Ultradent Products, Inc., South Jordan, Utah, USA) was used in the canals using a 25-size spreader (Mani Inc., Tochigi, Japan), followed by warm vertical condensation.
Scanning Electron Microscope Analysis: The specimens were longitudinally sectioned. Then samples were immersed in 17% EDTA solution for 10 minutes, immersed in 5.25 percent sodium hypochlorite for 10 minutes, and lastly washed entirely with water to remove and smear the layer. Carbon sputtering was performed in the middle and apical thirds of the root canal to aid SEM (500x magnification) evaluation.

[GUIDELINES]
To study the penetration of antioxidant agents within the dentinal tubules, scanning electron microscopy was used. (Figure 1) The mean and standard deviation of dentinal tubular penetration of EndoREZ sealer was assessed. Out of all the groups analyzed, group V (Grape Seed Extract) showed higher dentinal tubular penetration in both the middle third and apical third of the root canal with a mean value of 742.99μm and 460.21 μm respectively (p<0.05).Among all groups, group II (5.25% NaOCl+17%EDTA) showed the least dentinal tubular penetration at the middle third of the root canal (60.15μm) as well as in the apical third of the root canal (24.86μm).

[STEPS]
SECTION: Methodology
1. Extracted mandibular premolars (n=50) were taken. The samples were randomly divided into 5 groups. These samples were autoclaved, later decoronated using a sectioning disc to attain a 14-mm length of the root from the apex, segregated into five groups.To prevent apical extrusion, sticky wax was used to seal the apex of each root. Using an initial 10 K file (Dentsply Sirona, Ballaigues, Switzerland), the precise working length was determined, which was confirmed further by intraoral radiographs.
TITLE: Binding properties of  immunoglobulin-binding protein by double immunodiffusion (Ouchterlony) technique.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. The binding of SpLAG, SpLG and SpAG with animal sera, avian IgY,  and purified mammalian IgG are investigated by double immunodiffusion.
?. Briefly, 1% agarose gels are prepared and wells cut into the gel using a template.
?. Initially, aliquots of 25 µl each of the immunoglobulin-bacterial protein SpLAG, SpLG or SpAG at a concentration of one (1) µg/µl are applied to the centre well.
?. The peripheral wells are filled with 25 µl each of IgY (30 µg/µl) or mammalian IgG or sera.
?. The gels are incubated at RT for 48−72 hours.
?. After that the gels are examined for precipitin lines.
?. Human serum or human IgG are included as positive controls.
?. The positive results are taken as the presence of precipitin line/s and negative results, the absence of precipitin lines.
?.
TITLE: mPSM protocol
AUTHORS: Takehito  Tomita

[DESCRIPTION]
Protocol to perform 2D ex vivo segmentation assay (Lauschke et al. 2013).

[STEPS]
4. 
Extract mouse embryos and collect tails in dissection medium.


3. Wash the dried wells with ~200μL culture medium per well. Apply fresh culture medium (160uL ~ 300μL per well) and equilibrate in 37°C 5% CO2 until use.
2. Prepare dissection medium and culture medium.
1. Coat dishes/plates with fibronectin.
5. Cut the tail bud using a scalpel in dissection medium. Be careful not to flick the tail buds as this damages the tissue.
1.1. Dilute fibronectin (Sigma, Cat.No: F1141) with PBS to 50ug/mL (20 fold dilution).
1.2. Fill each well of 8-well plate (NuncTMLab-TekTMII Chambered Coverglass, Cat.No: 155409PK) with ~160μl of fibronectin solution and incubate for 1 hour in 37°C.
1.3. Remove all fibronectin solution from the well(s) and dry for 1 hour at room temperature (~25°C).
2.3. Add 510 µL to the remaining 30 mL mix in step 2.1. This mix is used as dissection medium. It can be kept at the bench until use.

2.2. Separate 10 mL from the mix in step 2.1 and filter through a 0.22μm filter (Millex-GV, Cat.No: SLGV033RS) into a fresh 15 mL falcon tube. Add 100 µL to achieve  
100U/mL. This mix is used as culture medium. Equilibrate in 37°C 5% CO2 until use.
2.1. Mix the following ingredients in a 50 mL falcon tube:

0.4 g 
400 µL 
35.2 µL 
40 mL 
Vortex well.
6. Wash the tail buds once in a dish containing culture medium. Quickly move the tail buds to the fibronectin coated wells. Align the tissue orientation using forceps to place the cut side downward.
7. Incubate in 37°C 5% CO2 for 1hr to let the tissue attach.


8. Place the well into the metal box to fit into the microscope. Add a strip of wet paper towel inside the metal box to prevent drying. Carefully move the box into the microscope, as tissue can detach quite easily.
9. Set up imaging.
TITLE: Time-lapse killing assay (spheroid - IncuCyte)
AUTHORS: Philippa R Kennedy, Peter Hinderlie

[DESCRIPTION]
Many tumors exist in vivo  as three-dimensional masses. In order to better model the dynamics of three-dimensional tumor growth and immune cell invasion, cancer cell lines are grown in low-adhesion plates that force them to interact with each other, forming a spheroid. After spheroids form in culture, immune effectors and drugs are added to the wells and their effect on the tumor mass is measured by transmitted light or fluorescence microscopy in a live-cell image system (IncuCyte, Sartorius Inc). 

[STEPS]
2. Resuspend target cells in their preferred media. Seed 2x104 target cells in 100 μL/well into a 96 well U-bottom low adhesion plate (ULA plate, Cat. No. 7007, Corning). 


3. Spin the plate at 40 g (500 RPM) in one orientation and then 80 g (700 RPM) in the opposite orientation without any brake applied to the centrifuge.
4. Capture an image of the plate using the live-cell image system (Incucyte S3 or Zoom, (Incucyte S3) to ensure the cells are in the center of the well. If cells are at the edges, resuspend them with a multichannel pipette (breaking up the pellet) and repeat the seeding, altering the centrifugal forces until a central pellet is achieved.
5. After allowing spheroids to form for 1-5 days, add effector cells and treatments on top of the spheroids. It is important to do this very slowly and precisely as the spheroids are not attached to the plate.
6. Replace in the plate in the live-cell image system and take images of the wells at regular intervals for 5 days.
1. Optional: If target cells that form a spheroid are to be themselves monitored by fluorescence, perform this labeling/transfection in advance (see Time-lapse killing assay - monolayer - Incucyte)
7. Image analysis is performed in the IncuCyte software. The size of each spheroid, as detected by edge analysis of the transmitted light image or fluorescence detection of the spheroid mass, is measured for each time point and normalized to the size of the spheroid at the time effectors and drugs were added (t = 0 h).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To extract plasmid DNA from <em>Agrobacterium</em>, to confirm the correct plasmid has been taken-up, this protocol allows you to directly perform a mini-prep on transformed <em>Agrobacterium</em>, digest this DNA and visualize it on a gel. This is without the need of rescuing the plasmid in <em>E. coli</em> or genotyping with PCR. This protocol using the commercially available Bioline ISOLATE II Plasmid Mini Kit, designed for use with <em>E. coli</em>. The standard protocol this is modified from is available here: https://www.bioline.com/au/downloads/dl/file/id/1219/isolate_ii_plasmid_mini_kit_protocol.pdf</p>

[BEFORE_START]
<p>Ensure RNase A has been added to Resuspension Buffer P1. Pre-warm the required amount of PW1 to 50-55 ºC before starting the extration. Ensure Ethanol has been added to PW2. </p>

[STEPS]
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