eamag/protocols-with-steps huggingface dataset:
TITLE: One-dimensional SDS-PAGE (9-18% TGX gel)
AUTHORS: Sigrid Verhelst

[DESCRIPTION]
Protocol for one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 9-18% TGX gel for visualization and quantification of histone proteins. 

[STEPS]
SECTION: Sample preparation
1. Dry samples (equal to 400.000 cells)

SECTION: Sample preparation
2. Resuspend samples in 10µl laemmli-buffer

SECTION: Sample preparation
3. Add 1µl β-mercaptoethanol to each sample
 
       
SECTION: Sample preparation
4. Vortex and spin down

SECTION: Sample preparation
5. Incubate for 7 minutes at 95°C in a thermoshaker

SECTION: Sample preparation
6. Spin down
SECTION: Prepare Criterion Cell
7. Place the criterion cell on ice in a fume hood

SECTION: Prepare Criterion Cell
8. Remove the sticker from the bottom of the gel cassette and check the gel for cracks

SECTION: Prepare Criterion Cell
9. Put the gel cassette in the criterion cell

SECTION: Prepare Criterion Cell
10. Fill the reservoir with running buffer (25mM Tris, 0.1% SDS, and 192mM glycine in MilliQ water) and take out the comb
SECTION: Running of the samples
11. Load the samples and standards (2 µg of bovine histones) on the gel (3 standards per gel: lane 1, lane 9 and lane 18)

SECTION: Running of the samples
12. Put the cover on the criterion cell 

SECTION: Running of the samples
13. Start running the gel on 200V

SECTION: Running of the samples
14. Stop running when the frontline is almost gone
SECTION: Visualization
15. Take out the cassette

SECTION: Visualization
16. Incubate in fixation-solution (7% acetic acid, 10% methanol in MilliQ water) for 10 minutes on a shaker

SECTION: Visualization
17. Wash the gel 3 times for 5 minutes in MilliQ water on a shaker

SECTION: Visualization
18. Incubate in SyproRuby overnight
  

SECTION: Visualization
19. Wash the gel 3x for 10 minutes in MilliQ water on a shaker

SECTION: Visualization
20. Visualize the gel (Versadoc)
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Zobell plates (1 L)</strong><br />15 g sea salts<br />1 g yeast extract<br />5 g peptone<br />fill to 1 L qH<sub>2</sub>O<br />14 g agar<br /><br /><strong>MLB (1 L)</strong><br />15 g sea salts<br />0.5 g yeast extract<br />0.5 g peptone<br />0.5 g casamino acids<br />3 ml glycerol<br />fill to 1 L qH<sub>2</sub>O<br /><br /><strong>MSM (1 L)&nbsp;</strong><br />450 mM NaCl (13.4 g)<br />50 mM MgSO<sub>4</sub>&middot;7H<sub>2</sub>O (6.16 g)<br />50 mM Tris base (3.02 g) OR 50 mL 1 M Tris (pH 7.6)<br />fill to 1 L qH<sub>2</sub>O<br />adjust to pH 7&ndash;8 if necessary<br /><br /><strong>Top MSM (top agar; 1 L)</strong><br />450 mM NaCl (13.4 g)<br />50 mM MgSO<sub>4</sub>&middot;7H<sub>2</sub>O (6.16 g)<br />50 mM Tris base (3.02 g) OR 50 mL 1 M Tris (pH 7.6)<br />fill to 1 L qH<sub>2</sub>O<br />adjust to pH 7&ndash;8 if necessary<br />6 g low melting point (LMP) agarose<br /><br />

[STEPS]
TITLE: Bogus Data Acquisition Protocol III
AUTHORS: Abby   Moore

[DESCRIPTION]
The purpose of this protocol is to demo protocol development.

[BEFORE_START]
Know before you start.

[GUIDELINES]
My guidelines.        

[STEPS]
2. I'll display an image using embed code retrieved from Box.


 

3. I'll show a command using the Command component:


 

1. Bogus Data Acquisition Protocol IBefore starting, you need to run a test using the following protocol. I referred to this protocol using a hyperlink: Bogus Data Acquisition Protocol I.
TITLE: Spinal manipulations for migraine
AUTHORS: Paul Posadzki, Edzard Ernst

[DESCRIPTION]
Several new randomized trials evaluating the effectiveness and safety of spinal manipulative therapies (SMT) have been published since our previous systematic review (SR). Therefore, we aim at updating the evidence base for the effectiveness of SMT for migraine. An update searches were performed in the following electronic databases: Amed, Embase, Medline, CINAHL, Mantis, Index to Chiropractic Literature, and Central using the exact same search terms that had previously been constructed over two concepts: SMT and migraine headache (without any methodological filters). In addition, bibliographies of all thus identified studies and relevant SRs were scanned for any relevant papers. We searched for English language papers but considered studies published in any language. 

[GUIDELINES]
We will follow the PRISMA 2020 guideline for reporting systematic reviews

[STEPS]
1. Spinal manipulations for migraine: a protocol for an updated systematic review and meta-analysis of randomized clinical trials
2. 
SECTION: Protocol
3.
TITLE: The Illumina libraries preparation for the Scapharca broughtonii
AUTHORS: Chang-Ming Bai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used to detail the process of Illumina libraries preparation for the</span><span style = "font-style:italic;"> Scapharca broughtonii</span><span> genome.</span></div></div>

[STEPS]
?. The extracted DNA was sheared into fragments about 350 bp in size using a Bioruptor Pico Sonication System (Diagenode, Seraing, Belgium), and verified by agarose gel electrophoresis.
The sizes of the main fragments should between 200 bp and 500 bp.
?. Fragments with size > 300 bp were purified using VAHTSTM DNA Clean Beads (Vazyme Biotech Co., Ltd, Nanjing, China).
?. Repaired using NEBNext® End Prep Enzyme Mix (NEB, E6091) and NEBNext® End Repair Reaction Buffer (NEB, B6052) keeped at 20℃, 30min; 65℃, 30min with the end-repaired fragments to obtain blunt ends which were then 3’-adenlyated to create sticky ends.
?. These DNA fragments were ligated at both ends to T-tailed adapters and amplified.
?. PCR was performed using NEBNext® Ultra™ II Q5® Master Mix (NEB, M0544), and the Index i7 and Universal i5 preimers. The temperature profile was 30 sec. at 98 ℃ followed by 10 cycles of 10 sec. at 98 ℃, 75 sec. at 65 ℃, and more 5 min. at 65 ℃ for further elongation.
INDEX i7 PRIMER: 5´-CAA GCA GAA GAC GGC ATA CGA GAT ATGACGTC GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC*T-3´NEBNext Universal i5 PCR Primer：5´-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC*T-3´
?. VAHTSTM DNA Clean Beads (Vazyme Biotech Co., Ltd, Nanjing, China) was used to purify the PCR production.
TITLE: Preparação para ir ao campo
AUTHORS: Alicia Rafaelly Vilefort Sales, Daniel Vartanian, Maria Augusta Medeiros Andrade

[DESCRIPTION]
Este protocolo foi desenhado para o processo de coleta de dados do projeto de pesquisa Sono e Gravidez. Consulte a aba Guidelines para visualizar o fluxograma do processo como um todo.

Caso tenha dúvidas ou sugestões, entre em contato com um dos autores.

---

This protocol was designed for the data collection process of the research project Sleep and Pregnancy. See the Guidelines tab to view the process flowchart as a whole.

If you have any questions or suggestions, please contact one of the authors.

[BEFORE_START]
Leia todo o procedimento antes de iniciá-lo;
Siga todas as etapas deste protocolo pelo menos 1 dia antes da data do recrutamento
Este protocolo assume que o(a) usuário(a) se encontra na sala do Grupo Interdisciplinar de Pesquisa em Sono (GIPSO). Os materiais citados neste documento já devem estar à disposição;
Entre em contato com a coordenação do projeto caso encontre algum problema.

[GUIDELINES]
O processo de coleta de dados deste projeto segue o seguinte fluxograma.

 
Com exceção da etapa Processamento e análise de dados, todas as etapas possuem protocolos próprios.

[STEPS]
SECTION: Agenda
1. Consulte a agenda do projeto e verifique a quantidade de gestantes a serem recrutadas em seu turno.

 

SECTION: Agenda
3. Verifique os horários e o trajeto que você terá que fazer para que você chegue com ao menos 30 minutos de antecedência do horário de sua primeira consulta na Casa de Parto.
SECTION: Actígrafos
4. Separe um actígrafo para cada gestante a ser recrutada e mais um actígrafo extra por segurança.
SECTION: Actígrafos
5. Realize o processamento de pré-coleta nos actígrafos com antecedência.

 


 

SECTION: Actígrafos
6. Armazene os actígrafos de maneira segura.

 

SECTION: Outros materiais
7. Separe os itens abaixo conforme a quantidade de gestantes que irá recrutar, sempre deixando alguns itens/cópias extras por segurança.

 

Termo de Consentimento Livre e Esclarecido  (TCLE);

 

 Formulário de campo;

 

Termo e recibo de entrega de actígrafo;

 

Formulário de controle;

 

1 Guia de instruções e cuidados com o actígrafo;

Saco plástico hermético de 35 x 45 cm para cada actígrafo

 

1 Braçadeira de 30 cm para cada voluntária

 

SECTION: Outros materiais
8. Separe também os seguintes itens:

1 Prancheta;
1 Porta crachá;
1 Folha de acesso;
1 Folha com os critérios de elegiblidade;
1 Cópia do protocolo de preparação para ir ao campo (este protocolo);
1 Cópia do protocolo de recrutamento de voluntárias;
3 Canetas esferográficas de cor azul ou preta.

 

SECTION: Aparelho celular
9. Certifique-se que você tem acesso à internet pela sua operadora de telefonia.

 

SECTION: Aparelho celular
10. Certifique-se que seu celular está com 100% de bateria.

 

SECTION: Ida ao campo
11. Siga para o recrutamento de voluntárias na casa de parto.

 

SECTION: Agenda
2. Confirme com a voluntária a ida dela para a consulta de abertura de plano (AP) um dia antes da data da consulta
TITLE: Data-Independent Acquisition (DIA) Data Processing using Spectronaut/directDIA (Biognosys): Whole Proteome Analysis of Human Whole Lung Tissue
AUTHORS: Joanna Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Proteolytic peptide measurement of Human whole lung tissue using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer for peptide/protein identification and quantification by processing DIA data using Spectronaut software.

[STEPS]
1. Search the DIA data with Spectronaut using the spectral library-free directDIA
algorithm against a Human UniProt-SwissProt proteome database (https://www.uniprot.org/proteomes), applying the settings described in Table 1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol refers to a study having the aim to identify and test at field artificial structures suitable as collectors for common cuttlefish Sepia officinalis eggs in wild condition.</p>
<p>During the same study, three different protocols were tested and compared. This one refers to the named 'seagrass' collector protocol, due to their similarity to the natural seagrass meadaw.</p>
<p>This work was supported by the project “Protection, improvement and integrated management of the sea environment and of cross-border natural resources – ECOSEA”, funded by EU Adriatic IPA Cross-border Cooperation 2007-2013 (2°ord./0236/0).</p>
<p> </p>

[BEFORE_START]
<p>An adequate vessel for transshipment/positioning and scuba divers are needed for a proper installation.</p>

[GUIDELINES]
<p>A total of 160 collectors were installed in the vertices of an electro-galvanized iron mesh having sheet size 2000 x 3000 cm, 15 x 15 cm mesh dimension and 0.6 cm diameter. The iron mesh was installed in a tubular galvanized frame of 4.83 cm diameter. A single eggs collector consist of 50 cm segments of polyethylene floating rope having a diameter of 0.8 cm. Each modulus has been installed on the seafloor at the beginning of the spawning period of the cuttlefish in a linear composition of 9 'seagrass' collectors. The modules have been tested in two central Adriatic Sea areas (Senigallia and Portonovo, Italy) where trawling is forbidden (by low and by a presence of anti-trawling structures). The structures were fixed on the seabed close to the artificial reefs modules in order to protect them from heavy storms.</p>
<p>This work was supported by the project “Protection, improvement and integrated management of the sea environment and of cross-border natural resources – ECOSEA”, funded by EU Adriatic IPA Cross-border Cooperation 2007-2013 (2°ord./0236/0).</p>
<p> </p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/5bn2xfe.jpg" data-src="https://s3.amazonaws.com/pr-journal/5bn2xfe.jpg" data-ofn="Seagrass nursery 3.jpg" /></p>
<p> </p>
<p> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/5di2xfe.jpg" data-src="https://s3.amazonaws.com/pr-journal/5dh2xfe.jpg" data-ofn="Seagrass collector.jpg" /></p>
<p> </p>
<p><video controls="controls" width="300" height="150"><source type="video/mp4" src="https://s3.amazonaws.com/pr-journal/5bp2xfe.mp4" /></video></p>

[STEPS]
?.
TITLE: Environmental DNA (eDNA) extraction with modified Qiagen DNeasy Blood & Tissue kit
AUTHORS: Collin Closek, Hilary Starks, Elizabeth Andruszkiewicz, Alexandria Boehm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nucleic acids extraction from the filters using the Qiagen DNeasy Blood and Tissue Kit with some modifications to the manufacturer’s protocol. </div></div>

[STEPS]
?. [Preparation]
Nucleic acids were extracted from the filters using the Qiagen DNeasy Blood and Tissue Kit with some modifications to the manufacturer’s protocol.
?. [Preparation]
Prior to extraction, 0.5 mm and 0.1 mm glass beads (BioSpec Products) transferred to glass beaker, covered with tin foil, and ashed at 500 °C for 5 hours.
?. [Preparation]
0.75 g of each bead size was distributed into sterile 5 ml extraction tubes, the tubes with caps loosened were subsequently autoclaved and UV treated for 30 min.
?. [Extraction]
To serve as a control in each set of extractions, an additional empty tube with beads was carried through the process as an extraction blank.
?. [Extraction]
Ashed beads were added to sample filters tubes with subsequent bead beating steps as in Djurhuus et al., 2017, and then incubated at 56 °C overnight with 900 μl Buffer ATL and 100 μl Proteinase K (Qiagen).
?. [Extraction]
Bead free supernatant was transferred to 2 ml tube, 650 μl Buffer AL was added, and then briefly vortexed. An additional 650 μl Ethanol (96-100%, molecular grade) was added, then vortexed briefly.
?. [Extraction]
Mixture was added into mini spin column (650 μl, 3x). Remaining steps followed the manufacturer’s protocol for the DNeasy Blood and Tissue Kit.
?. [Extraction]
Samples were subsequently diluted 1:10 for 12S rRNA gene amplicon and metabarcoding preparation.
TITLE: Pollen germination on wheat stigmas 
AUTHORS: Marina Millán Blánquez

[DESCRIPTION]
Aniline blue staining of pollen tube growth on the wheat stigma and through the style 4 h and 30 min after pollination.

[STEPS]
SECTION: Hand pollination
1. Please refer to wheat-training.com for detailed explanations on how to perform hand pollinations. Link to pdf: https://www.wheat-training.com/wp-content/uploads/Wheat_growth/pdfs/How-to-cross-wheat-pdf.pdf
SECTION: Carpel dissection and fixation
2. Using a pair of tweezers, dissect carpels 4.5 h after pollination to allow sufficient time for pollen tube
emergence. 
SECTION: Carpel dissection and fixation
3. Store samples in a fixative solution of 95% ethanol and absolute acetic acid (75% v/v) and kept at 4 °C until sample preparation for fluorescence microscopy.
SECTION: Aniline blue staining of pollinated stigmas
4. On the day of sampling, prepare a solution of 0.1% aniline blue in 0.1 M K3PO4. You can prepare a stock solution of 1% aniline blue dissolved in 1x PBS. The stock solution should be kept in the fridge at
4 °C. Use tin foil to avoid exposure to light. 
SECTION: Aniline blue staining of pollinated stigmas
5. Wash fixed samples three times for 5 minutes in sterile water and transferred to 0.1% aniline blue
solution and kept overnight at 4 °C. Use tin foil to avoid exposure to light. 
SECTION: Aniline blue staining of pollinated stigmas
6. Without washing the samples, dissect out the ovary using a sharp razor blade. Try not to damage the stigma. Leave the remaining stigmatic tissue to dry at 45 °C in a hot plate for a few minutes until most of the aniline blue solution is evaporated. Cover the hot plate with an opaque lid to avoid exposure to light. 
SECTION: Aniline blue staining of pollinated stigmas
7. Use Vectashield (catalogue No. H-1000-1, 2BSCIENTIFIC LTD) as an antifade mounting media to
preserve fluorescence. 
SECTION: Aniline blue staining of pollinated stigmas
8. Happy microscopy.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Efficiency of plaquing (EOP) assay for <em>C. jejuni </em>phages</p>
<p>Jessica Sacher (with help from Stephanie Guerra</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Enzymatic disaggregation of human myometrium for 10X Single Cell RNA-seq
AUTHORS: Aymara Mas, Alba Machado-Lopez, Patricia Escorcia, Carlos Simón

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the procedure for dissociating myometrial samples, which is based in an enzymatic disaggregation using Collagenase IV and DNAse I. This protocol is adapted from Mas et al. 2012 </span><span style = "font-style:italic;">Fertil Steril</span><span> with minor modifications. </span></div></div>

[STEPS]
?. [Tissue collection and transportation]
Uterine tissues should be transported in preservation solution (HypoThermosol® FRS) at  from the surgery room to the lab.
4 °C
?. [Dissociation]
Rinse the sample with wash buffer, removing blood or mucus.
?. [Remove red blood cells]
[Optional] If after centrifugation there is a ring of red blood cells, add  ACKL lysis buffer, consisting in hypotonic shock, and incubate at  for . Then, follow step 7 again.
37 °C
?. [Dissociaton]
Add  Tryple Select Enzyme to resuspended media containing cells and mix thoroughly by pipetting. Incubate for    -  at  and centrifuge at .
400 µl
37 °C
Centrifuge: 300 33
?. [Prepare cells for chromium]
Count the total number of cells (at least twice on two different cell counters).
?. [Dissociation]
Place wet tissue under a petri dish. Isolate with a sterile scalpel the myometrial layer by removing the endometrium, serosa and laser-burnt zones.
?. [Dissociation]
Mechanical disaggregation by chopping and thoroughly mincing up the tissue into small pieces (3).
?. [Dissociation]
Transfer contents to 50 ml falcon tubes containing  of enzyme buffer. Tighten lid, seal with parafilm and incubate at  overnight horizontally.
30 ml
37 °C
?. [Dissociation]
Afterwards, cell suspensions should be filtered through 100 to 50-μm polyethylene filters to remove cellular clumps and undigested tissue.
?. [Dissociation]
Centrifuge the filtered medium  .  Remove supernatant and add at least  of resuspension buffer (depending of the pellet content).
Centrifuge: 400 34, 5 min
1 ml
?. [Dissociaton]
Add  DNAse I to digest the extracellular genomic DNA and mix thoroughly by pipetting. Incubate for at.
100 µl
37 Room temperature
?. [Dissociaton]
Add at least  of resuspension buffer. Filter through a 40 μm cell strainer and centrifuge at .
1 ml
Centrifuge: 400 34, 5 min
?. [Dissociaton]
Remove supernatant and resuspend the content in at least  of resuspension buffer (depending of the pellet content).
1 ml
?. [Prepare cells for chromium]
Proceed to 10x experiment.
?. [Prepare cells for chromium]
[Optional] Resuspend cells in freezing medium to a concentration of 1 x 106 cells for storage in cryogenic vials.
TITLE: Human Neurosurgical Tissue Processing
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for receiving human surgical tissue, followed by trimming, mounting, embedding, and slicing under sterile conditions in order to produce tissue suitable for both slice culture as well as acute slice electrophysiology recording.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE:  Addgene Covid-19, Back To Work Training
AUTHORS: Joanne Kamens, Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Disclaimer</span><span>: The SOP presented here has been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene 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: Parhyale hawaiensis embryo single cell dissociation
AUTHORS: Leo Blondel

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

[STEPS]
?. [Coat all gear with BSA]
Prepare 1% BSA solution in ddH2OCoat 1 or more wells from 12 well plateCoat pasteur pipettes for embryo transfersIf using syringes for loading coat tubing and syringes
?. [Collect embryos]
This protocol was tested on embryos at 12hpf to 72hpf.From the collected females, harvest embryos in the pouch and place them in FASW.
?. [Remove Egg Shell]
Place 30 embryos on a Sylgard plate or a 2% agar in FASW plate in a small drop.Aspirate as much as possible of the FASWWash with a drop of dissociation buffer. Repeat until no precipitate is visible.Using the tungsten needles, remove the egg shell around the embryos. Make sure to place the egg shells away from the embryos as they will prevent the correct dissociation.Once all embryos have been separated from the egg shell, transfert them into the well filled with dissociation buffer.
?. [Prepare material]
12 well plateTungsten needlesParafilm, cut into squares a little bigger than the diameter of the well.TapeElmer's paste or silicon seal
?. [Prepare dissociation buffer and PBS Optiprep]
Prepare dissociation buffer, for 40ml in ddH2O:  ABC1quantityunit2Isethionic acid sodium600mg3Sodium pyrophosphate360mg4CAPS88mg5ddH2O40ml6Total40mlPrepare 2ml PBS optiprep solutions, all volumes in ul  ABCDE15%10%20%30%210x PBS2002002002003ddH2O17001600140012004Optiprep100200400600
ABC1quantityunit2Isethionic acid sodium600mg3Sodium pyrophosphate360mg4CAPS88mg5ddH2O40ml6Total40ml
ABCDE15%10%20%30%210x PBS2002002002003ddH2O17001600140012004Optiprep100200400600
?. [Work a 4C for the whole protocol.]
For the whole protocol, work at 4C, and place all solutions on ice.
?. [Prepare the dissociation well]
Remove the 1% BSA from the wells of the 12 well plate and wash with dissociation buffer once. The fill halfway with the dissociation buffer.On the lid, locate the position of that well and place a ring of elmer''s paste that will serve as seal.Place tape on the edges of the lid to allow for it to stay in place once closed.
?. [Prepare the well for dissociation]
Fill the well with the embryos all the way with dissociation buffer until a meniscus is visible. Place the parafilm square on the meniscus. THERE MUSN'T BE ANY BUBBLES TRAPPED.Close the lid, sealing the parafilm in place thanks to the paste, and fix it with the tape around.
?. [Dissociate the cells]
?. [Clean the dissociation buffer]
Remove the plate from the vortex and let the cells settle for 5 minutesOpen the lid and assess that the dissociation worked.Aspirate 1ml of liquid at the top of the wellThen fill from the bottom with 400 ul of 5% Optiprep PBSThen 200ul of 10% optiprepThen 100ul of 20% optiprepAnd finally 300ul of 30% optiprep
?. [Centrifuge to concentrate the cells at the 20% mark]
Centrifuge the plate at 2500 RPM for 3-5 minutes to concentrate the cells at the margin.
?. [Collect the cells]
Using the BSA coated syringe, aspirate the cells collected at the 20% optipre band.