eamag/protocols-with-steps huggingface dataset:
TITLE: GeoMx Digital Spatial Profiler (DSP) Protocol - University of Minnesota TMCs 
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
The nanoString GeoMx‱ Digital Spatial Profiler (DSP) is a platform that allows high-plex profiling at the protein and RNA level, providing spatial and temporal assessment of tumors in frozen or formalin-fixed paraffin-embedded limited tissue sample.

The following protocols/manuals were used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. 

[STEPS]
SECTION: Slide Preparation
1. GeoMx DSP Manual Slide Preparation User Manual
  

SECTION: ROI Acquisition
2. GeoMx DSP Instrument User Manual
  

SECTION: Library Preparation
3. GeoMx DSP NGS Readout User Manual
  
 


SECTION: FASTQ Generation
4. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq version 2.20.0
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A real-time PCR to detect Orientia tsutsugamushi DNA. This method has been adapted from a publication by Jiang et al 2004, and  the oligonucleotides have been modified and a different PCR kit used.</p>

[GUIDELINES]
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol.</li>
</ul>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Trajectories of Glucocorticoid-Therapy  After Initiation in Early (or Methotrexate-Naive) Rheumatoid Arthritis:  Protocol for a Systematic Review, Meta-Analysis and Meta-Regression of  Observational Cohort Studies
AUTHORS: Andriko Palmowski, Frank Buttgereit

[STEPS]
?. [Objective]
To assess real-world trajectories of glucocorticoid-therapy initiated as part of early rheumatoid arthritis (RA) treatment.
?. [Eligibility]
Observational cohort studies (CS) published between Jan 01, 2005 and now. Studies must enroll early (or methotrexate-naive) RA patients and report on those starting/taking systemic glucocorticoids (GCs) at baseline and describe at least one of our outcomes at two time points minimum.
?. [Data Source]
MEDLINE (via PubMed).
?. [Search Strings]
Search strings were developed utilizing the PICOS mnemonic:Population: early (or methotrexate-naïve) RA - Intervention: systemic GCs - Comparator: any - Outcome: any - Study design: observational cohort study
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							(Rheumatoid[Title] OR Arthritis, Rheumatoid[MeSh:NoExp]) AND (early[title/abstract] OR naïve[title/abstract]) AND (Cortison*[title/abstract] OR Predniso*[title/abstract] OR Cortico*[title/abstract] OR Glucocort*[title/abstract] OR Steroid*[title/abstract] OR Glucocorticoids[MeSh:NoExp]) AND (Cohort[Title/Abstract] OR Longitudinal[Title/Abstract] OR Prospective[Title/Abstract] OR Registry[Title/Abstract] OR Longitudinal Studies[Mesh:NoExp] OR Cohort Studies[Mesh:NoExp] OR Registries[Mesh:NoExp]) AND 2005/01/01:2021/01/01[dp] NOT (random*[Title] OR randomized controlled trial[pt])NOT (review[Title] or review[pt])
?. [Outcomes]
Mean/median dose of prednisone equivalent, proportion of patients who could discontinue GCs, proportion of patients who could reduce GCs (all: at baseline, 3, 6, 9, 12, and 24 months).
?. [Data Synthesis]
Meta-analysis will be conducted to pool results, including meta-regression (mixed effects) to assess change over time for each outcome.
TITLE: Transfection of construct containing kinetoplastid Blastocrithidia sp. p57 UTR's in Diplonema papillatum using AMAXA Nucleofactor apparatus.
AUTHORS: Binnypreet Kaur

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Transfection of construct containing kinetoplastid </span><span style = "font-style:italic;">Blastocrithidia</span><span> sp. p57 UTR's in </span><span style = "font-style:italic;">Diplonema papillatum</span><span> using AMAXA Nucleofactor apparatus.</span></div></div>

[STEPS]
TITLE: LC-MS/MS Untargeted Metabolomics Data Processing
AUTHORS: Kevin Contrepois

[DESCRIPTION]
Scope:
To describe the procedure to process untargeted metabolomics data.

Expected outcome/data:
Metabolomics dataset is processed using commercial software and custom R scripts. Metabolite annotation was performed using an in-house library and spectral databases.

[STEPS]
1. Data from each mode were independently analyzed using Progenesis QI software (v2.3) (Nonlinear Dynamics, Durham, NC). 
2. Metabolic features from blanks and those that didn’t show sufficient linearity upon dilution in QC samples (r < 0.6) were discarded. 
3. Only metabolic features present in >2/3 of the samples were kept for further analysis. 
4. Median normalization was applied to correct for differential starting material quantity. 
5. Missing values were imputed by drawing from a random distribution of low values in the corresponding sample. 
6. Data from each mode were merged and metabolites were formally identified by matching fragmentation spectra and retention time to analytical-grade standards when possible or matching experimental MS/MS to fragmentation spectra in publicly available databases. 
7. Metabolite abundances were reported as spectral counts.
8. Peak annotation was first performed by matching experimental m/z, retention time and MS/MS spectra to an in-house library of analytical-grade standards. Remaining peaks were identified by matching experimental m/z and fragmentation spectra to publicly available databases including HMDB (http://www.hmdb.ca/), MoNA (http://mona.fiehnlab.ucdavis.edu/) and MassBank (http://www.massbank.jp/) using the R package ‘MetID’ (v0.2.0) (PMID: 30944337). Briefly, metabolic feature tables from Progenesis QI were matched to fragmentation spectra with a m/z and a retention time window of  ±15 ppm and ±30 s (HILIC) and ± 20 s (RPLC), respectively. When multiple MS/MS spectra match a single metabolic feature, all matched MS/MS spectra were used for the identification. Next, MS1 and MS2 pairs were searched against public databases and a similarity score was calculated using the forward dot–product algorithm which takes into account both fragments and intensities (PMID: 24222034). Metabolites were reported if the similarity score was above 0.4. Spectra from metabolites of interest were further manually investigated to confirm identification.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the basic steps for a reciprocal best hit BLAST. It will show you how to download sequences from NCBI, creating BLAST databases, and how to blast.</p>

[GUIDELINES]
<p>I recommend running this protocol in a docker container, so you don't have to worry about installation.</p>
<p> </p>
<p>Use this docker container: https://hub.docker.com/r/biodckr/blast/</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Rine Lab Reopening SOP
AUTHORS: Rine Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a living document that should reflect our current understanding of best practices. It is subject to change and will evolve as the situation changes and our understanding evolves.</div><div class = "text-block"><span style = "font-weight:bold;">Disclaimer</span><span>: The SOP presented here has been designed by the Rine Lab at University of California, Berkeley and is being shared outside of UC Berkeley for informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>, can be held responsible for your use of the information contained in or linked to from these web pages.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Reference:&nbsp; Fuhrman&nbsp;et&nbsp;al,&nbsp;1988. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC202673/" target="_blank">Extraction from Natural Planktonic Microorganisms of DNA Suitable for Molecular Biological Studies</a>.&nbsp;AEM&nbsp;54(6),&nbsp;1426‐1429

[GUIDELINES]
<strong>modified by Rohan Sachdeva, 08132010; Cheryl Chow 03092011<br /><br />Supplies:<br /></strong> a)&nbsp;Dry&nbsp;ice <br />b)&nbsp;15&nbsp;ml&nbsp;falcon&nbsp;tube&nbsp;&ndash; 1&nbsp;per&nbsp;filter <br />c)&nbsp;Oak&nbsp;Ridge&nbsp;tube&nbsp;&ndash; 1&nbsp;per&nbsp;filter;&nbsp;Nalgene&nbsp;#3119-ÃÂÃÂ0050&nbsp;(polypropylene) &nbsp;<br />d)&nbsp;2&nbsp;x&nbsp;2ml&nbsp;NON&nbsp;lo-ÃÂÃÂbind&nbsp;tube/filter&nbsp;for&nbsp;1<sup>st</sup> and&nbsp;2<sup>nd</sup> DNA&nbsp;precipitation&nbsp;steps&nbsp;(Day&nbsp;2 steps 4,&nbsp;14) <br />e)&nbsp;2&nbsp;x&nbsp;1.5ml&nbsp;lo&acirc;ÃÂÃÂbind&nbsp;tube/filter&nbsp;(Day&nbsp;3, step 6) <br />f)&nbsp;1&nbsp;x&nbsp;PCR&nbsp;tube/filter&nbsp;for&nbsp;2ng/&mu;l&nbsp;dilution <br />g)&nbsp;pH&nbsp;paper,&nbsp;range 5.5&acirc;ÃÂÃÂ9<br /><br /><strong>Reagents: &nbsp;</strong>&nbsp;Make&nbsp;all&nbsp;solutions&nbsp;with&nbsp;autoclaved&nbsp;0.02&micro;m&nbsp;filtered&nbsp;MilliQ&nbsp;water&nbsp;and&nbsp;autoclave. <br />a)&nbsp;10X&nbsp;STE:&nbsp;100mM&nbsp;Tris-Cl&nbsp;(pH&acirc;ÃÂÃÂ8.0),&nbsp;1M&nbsp;NaCl,&nbsp;and&nbsp;10mM&nbsp;EDTA).&nbsp;Dilute&nbsp;to&nbsp;1X&nbsp;STE.&nbsp;<br />b)&nbsp;10%&nbsp;SDS:&nbsp;do&nbsp;NOT&nbsp;autoclave. <br />c)&nbsp;10.5M&nbsp;NH<sub>4</sub>OAC <br />d)&nbsp;100%&nbsp;Ethanol&nbsp;(200&nbsp;proof,&nbsp;molecular&nbsp;grade) <br />e)&nbsp;1X&nbsp;TE,&nbsp;pH&nbsp;8.0:&nbsp;10mM&nbsp;Tris-ÃÂÃÂCl (pH&nbsp;8.0)&nbsp;and&nbsp;1mM&nbsp;EDTA <br />f)&nbsp;Phenol,&nbsp;pH&nbsp;8 <br />g)&nbsp;SEVAG&nbsp;aka&nbsp;chloroform:&nbsp;isoamyl&nbsp;alcohol <br />h)&nbsp;Phenol:&nbsp;chloroform:&nbsp;isoamyl&nbsp;alcohol,&nbsp;pH&nbsp;8&nbsp;(optional)<br /><br /><strong>Day 1 Note:</strong>&nbsp;**Process&nbsp;in&nbsp;sets&nbsp;of&nbsp;4&nbsp;samples&nbsp;through&nbsp;step&nbsp;#9 and&nbsp;spin&nbsp;all&nbsp;together.<br /><br /><strong>Reference:</strong>&nbsp; Fuhrman&nbsp;et&nbsp;al,&nbsp;1988.&nbsp;Extraction&nbsp;from&nbsp;Natural&nbsp;Planktonic&nbsp;Microorganisms&nbsp;of&nbsp;DNA&nbsp;Suitable&nbsp;for Molecular&nbsp;Biological&nbsp;Studies.&nbsp;AEM&nbsp;54(6),&nbsp;1426-ÃÂÃÂ1429

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: (NANOPORE MENU) Protocols for SARS-CoV-2 Bioinformatic Analysis and Database Submission
AUTHORS: Technical  Outreach and Assistance for States Team

[DESCRIPTION]
This collection of protocols covers library preparation and sequencing on Oxford Nanopore platforms as well as bioinformatic analysis and submission of SARS-CoV-2 data. These protocols have been assembled by the Technical Outreach and Assistance for States Team (TOAST) and the Office of Advanced Molecular Detection (OAMD) at the Centers for Disease Control and Prevention for use by participating State, Tribal, Local and Territorial labs for sequencing of SARS-CoV-2. Individual protocols were developed by the protocol authors, and individual protocols should be credited accordingly. 

These documents are intended for laboratories designated by TOAST and should not be widely or openly disseminated.

  


For technical assistance, please contact: TOAST@cdc.gov



[STEPS]
TITLE: Sterilization of ISE
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sterilization of ISE</div></div>

[STEPS]
?. [Sterilization]
Standard cleaning is best done with DI water.If sterilization is needed, use ethyl alcohol (typically >90%) or isopropyl alcohol (typically 60-70%) and swirl in the solution for ~10 seconds.
?. [Sterilization]
Then rinse vigorously with DI or tap water and recondition in the applicable analyte Standard Solution.
We do NOT recommend using household or other bleach on ISEs.
TITLE: PCR (Instructor Protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for the CURE's two PCRs,
 

and 
 

The URA3 PCR is preparative, to make the URA3 cassette for the gene knockout. The knockout PCR is diagnostic, to see whether the knockout succeeded.

[STEPS]
SECTION: Setup
1. Aliquot    into 100 µL aliquots, 1 per 4 students.

SECTION: Setup
2. If not done already, aliquot   into 1 mL aliquots, 1 per 4 students.

SECTION: Setup
3. For the URA3 PCR: grow and miniprep the E. coli hosting YTK74, using   or similar. Dilute to 1 ng/ul in elution buffer and make  20 µL aliquots, 1 tube for every 4 students.

SECTION: Instructor Tips &amp; Common Student Errors
4. Instructor Tips
Students often struggle with PCR. It's tetchy - I still struggle with it occasionally! Several choices make success more likely:
    - Hot-start Q5 master mix. The Q5 master mix is really robust, and the hot-start formulation keeps the enzyme from chewing up primers in the wait between mixing the reaction and starting it in the thermocycler.
    - For the URA3 preparative PCR - purified plasmid template. No nuclease contamination from suboptimal genomic DNA preps.

The idea that PCR works by an exponential DNA amplification is another thing that students often struggle with. I firmly believe that students should understand why they're doing what they're doing instead of just following a protocol. How best to motivate and evaluate student understanding is something I'm still struggling with.

Related to the above, the fact that there are three sets of oligos -- and that they're used in different ways, for different protocols -- is another thing to help students with. Why can't you just reuse the targeting oligos for PCR?

There is a LOT of pipetting of small volumes here. A reminder of good small-volume pipetting technique would not go amiss.

Interpreting the PCR gel often helps students solidify their understanding of what's going on here. Why is it important to estimate the PCR amplicon's size, not just see whether there's DNA there or not? For the knockout PCR, what does a long amplicon vs a short amplicon tell us?

SECTION: Instructor Tips &amp; Common Student Errors
5. Common student errors
Used wrong primers or template
Pipetting errors!
TITLE: Efficacy of Electrical Stimulation (ES) on Upper Limb Neurological and Functional recovery in Patients with Spinal Cord Injury: Protocol for a Randomised Controlled Trial
AUTHORS: Md Obaidul Haque, Hassan M. Al- Emran

[DESCRIPTION]
Background: Spinal cord injury (SCI) causes physical disability including a deterioration of upper limb function that have a considerable impact on an individual’s functional status and independence. Electrical stimulation (ES) is the use of low-energy electrical pulses to induce muscular contraction, particularly in SCI patients. This study will determine the ES efficacy for upper limb functional and neurological improvement in SCI patients.
Methods: Forty traumatic tetraplegic (TT) SCI patients will be recruited to this single blind clinical trial and will be randomly allocated to intervention and control group. The intervention group will receive a one-hour treatment that includes ES for 30 minutes and rest of the usual therapy for 4 weeks.The primary outcomes are Capabilities of Upper Extremity (CUE) Instruments, ASIA impairment scale (ASIA motor scores, ASIA sensory scores and neurological level). The secondary outcomes include Modified Ashworth Scale (MAS) and Manual muscle testing (MMT). ES will be placed on the extensor and flexor group of muscles of the wrist and elbow. Baseline will be performed at the commencement of participant recruitment in the trial and post-test will be performed after ten sessions and consequently twenty sessions of the intervention. The recruitment of participants started on October 1st, 2022, and the trial will be continued until the completion of recruitment of forty participants. 
Analysis: Data will be analysed based on the nature of data. Categorical data will be displayed using frequencies and corresponding percentages, whereas continuous data will be displayed using descriptive statistics. Intention-to-treat analyses will be carried out. 
Ethics and dissemination: The study has been evaluated by the Ethics Committee of Centre for the Rehabilitation of the Paralysed and Jashore University of Science and Technology, Jashore, Bangladesh. All participants will provide consent. The protocol will follow the SPIRIT (Standard Protocol Items for Randomized Trials) statement. All protocol requirements will be followed during the duration of the trial. Findings will be disseminated in peer- reviewed publications and presented at academic, national and international conferences.
Trial registration number: [CTRI/2021/11/038271].

[STEPS]
TITLE: Bacteria Staining
AUTHORS: Guillermo Fernández Rodríguez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A bacteria staining protocol has been automated by OT-2. It allows to check the amount of target we had coated on the 96 well plate. </div><div class = "text-block">(We used 5 replicates per dilution)</div></div>

[STEPS]
?. [Staining bacteria ]
Inoculate a single colony of E.Coli DH5α from LB agar plate in   of LB. Use a sterile pipette tip, selecting a single colony from LB agar plate. The liquid culture is incubated overnight .
10 ml
37 °C
?. [Staining bacteria ]
Spin at   for  . Discard the supernatant, collect pellet and re-suspend in   of NaHCO3-Na2CO3, 50mM, pH 9,6. Mix by inverting the tube.
Centrifuge: 4000 33
10 ml
?. [Staining bacteria ]
Spin at 4000 rpm for   Discard the supernatant, collect pellet and re-suspend in  of NaHCO3-Na2CO3, 50mM, pH 9,6. Mix by inverting the tube.
8 ml
?. [Staining bacteria ]
Read the absorbance (600nm). Dilute the sample with NaHCO3-Na2CO3, 50mM, pH 9,6. and adjust the absorbance to 1.
?. [Staining bacteria ]
Make the following dilutions with -Na2CO3, 50mM, pH 9,6:1:5, 1:10, 1:30, 1:50 and 1:100.
?. [Staining bacteria ]
Add  the sample into 96 well-plate Nunc MaxiSorp. Incubate overnight at  .
200 µl
4 °C
?. [Staining bacteria ]
Wash 3x  PBS Tween 0,1%, pH 7,4.  Remove the drops after the last wash.
200 µl
?. [Staining bacteria ]
Add   of crystal violet/well. Incubate for  at
150 µl
37 Room temperature
?. [Staining bacteria ]
Wash 4x  destilled water. Wait for   for air-drying before counting colonies in the microscope.
250 µl
TITLE: USDA LTAR Common Experiment measurement: Cereal crop mycotoxin concentration
AUTHORS: Brook J. Wilke, Martin I. Chilvers

[DESCRIPTION]
Crop pests and diseases can reduce crop yields, but they can also worsen crop quality via reduced grain density or accumulation of toxins produced by fungal diseases. Several mycotoxins are known to harm crop quality, with deoxynivalenol (a.k.a. Vomitoxin or DON) being one of the most common, especially in corn and cereal grains. Subsequently, deoxynivalenol concentration can be correlated with disease severity. This protocol describes a process for measuring DON in cereal grains harvested from LTAR plots and fields.

[STEPS]
SECTION: Sample collection, processing, and analysis
1. Collect a representative subsample of grain from each experimental unit in the study that contains grains produced by grass species possibly infected with DON, including plots and fields. 
 

SECTION: Sample collection, processing, and analysis
2. If using the whole plot/field harvest, combine multiple subsamples from each plot or field to accurately represent the entire plot or field.
SECTION: Sample collection, processing, and analysis
3. Dry grain samples to a stable moisture content and then submit them to a laboratory for analysis of deoxynivalenol (a.k.a. Vomitoxin or DON).
SECTION: Sample collection, processing, and analysis
4. Sites may choose to submit samples to a commercial laboratory or analyze them on-site.
SECTION: Sample collection, processing, and analysis
5. Several devices are available for on-site DON testing. For on- site analysis, follow the protocol suggested by the manufacturer of your device .
SECTION: Covariate metrics to be sampled concurrently
6. Other mycotoxins may be quantified at the same time, including aflatoxin, ochratoxin, zearalenone, fumonisin, or ergot alkaloid.
SECTION: Covariate metrics to be sampled concurrently
7. Analyze non-grass crops for other mycotoxins that are expected and specific to that crop.
SECTION: Covariate metrics to be sampled concurrently
8. Note observations of diseases and pests, including presence and abundance, by crop scouting
exercises. These data will be important covariates to consider when evaluating mycotoxin
concentrations.
SECTION: Calculations
9. Determine mycotoxin levels as a concentration (e.g., ppm) and report them in the database using those units.
SECTION: Quality assessment
10. Be sure to dry grain to stable moisture quickly after harvest and store at Room temperature until
analysis.
SECTION: Quality assessment
11. Representative subsamples are critical from each experimental unit analyzed.
SECTION: Archiving
12. Grain samples should already be archived based on the crop productivity protocols.

 

SECTION: Recommendations for data collection
13. Table 1. Summary of recommendations for measuring mycotoxins.
TITLE: Isolating human intestinal crypts from biopsies for organoid generation
AUTHORS: Ran Zhou, Candace Cham, Jason Koval

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides details on crypt isolation from human terminal ileum and colon to robustly generate organoids.</div></div>

[STEPS]
?. Collect biopsies in PBS or culture media in 1.5 ml Eppendorf tubes.
?. Incubate all the tissues from each biopsy in 25mL of 8 mM EDTA/PBS in a 50 ml conical tube for 30 min, rocking (not shaking) at 4C.
4 °C
?. Place one 100 um cell strainer on each well of a 6-well plate. Pre-wet the cell strainer with 1mL culture media.
?. Transfer biopsies from EDTA/PBS into 1-2 ml ADF.
?. Using a customized P1000 aerosol resistant tip, pipet up and down vigorously, making sure that the biopsy tissue goes in and out of the tip, but with some difficulty. This helps release the crypts.
Customize the pipet tip: tips can be altered by cutting it to slightly smaller than the size of the largest biopsy, using a sterilized razor blade.  Cut the pipet tip at a straight angle, not biased, to keep the hole as small as possible.
?. Use the 100 um cell strainer to filter the tissue and large pieces from the crypts. Collect the crypts into a 1.5 ml microfuge tube. Check to see whether enough crypts were released from the tissue on a microscope.
?. Centrifuge the crypts at 300-400 g at 4C for 5 min in a tabletop swinging bucket centrifuge
Centrifuge: 300 34, 5 min
4 °C
A microcentrifuge is not recommended here because it is a fixed angle and we want the pellet to be at the bottom of the tube, not on the side
?. Remove the supernatant and resuspend the crypts in pre-warmed and CO2-equilibrated human organoid media (complete ADF) containing growth factors
?. Mix crypts with thawed matrigel in a ratio of 1:2 (cells:matrigel). Pipet up and down; avoid creating bubbles
?. Plate crypt/matrigel mixture onto a pre-warmed 12- or 24-well tissue culture plate, 100 or 50 ul/well respectively.
?. Incubate 45-60 min in 37C, 5% CO2 incubator
37 °C
[Incubation]
?. Add complete ADF into each well (24-well: 0.5 ml/well; 12-well: 1 ml/well)
?. Feed organoids with complete ADF every other day. Also add ciprofloxacin (10 ug/ml) to make sure that there are no remaining bacteria in your culture.
Media is changed every other day. Organoids are split and expanded as needed, about once per week.
?. Add fresh growth factors every 2-3 days and change complete ADF media as needed (approximately once per week or when media begins to change color).
?. Once biopsies received, wash 3 times with 25 ml ice cold PBS each in 50 ml conical tubes, or until PBS looks clear.
Biopsied tissues range from 6 to 20 mm3 in size. All the tissues biopsied from the same site are pooled for downstream procedures.
?. Prechill PBS and 8mM EDTA in PBS on ice. Pre-warm human organoid medium and 12-well/24-well plate in the tissue-culture incubator.