All murine 2D colony-forming assays were performed using female FVB wild-type mice. In brief, total dissociated mammary cells or FACS-purified basal and luminal cells were seeded together with irradiated NIH 3T3 cells. Cells were allowed to adhere, and either vehicle control (0.1% DMSO) or the indicated concentrations of epigenomic inhibitor were added. Cells were cultured for 7 d at 5% oxygen to allow basal colony growth in either Epicult-B mouse medium (Stem Cell Technologies) supplemented with 5% FBS, 10 ng/ml EGF, 20 ng/ml basic FGF, 4 µg/ml heparin, and 5 µM ROCK inhibitor (Millipore) or DMEM/F12 (1:1) supplemented with 10% FBS, 5 µg/ml insulin (Thermo Fisher Scientific), 10 ng/ml EGF, 10 ng/ml cholera toxin, 1.8 × 104 M adenine (Sigma), 0.5 µg/ml hydrocortisone, and 10 µM ROCK inhibitor (Millipore). Growth factors and hydrocortisone were obtained from Stem Cell Technologies.
Rock inhibitor
Manufactured by Merck Group
Sourced in United States
ROCK inhibitor is a type of laboratory equipment used for research and experimentation. It functions to inhibit the activity of the Rho-associated protein kinase (ROCK) enzyme, which plays a role in various cellular processes. The core function of the ROCK inhibitor is to provide a tool for researchers to study and manipulate ROCK-related mechanisms in a controlled laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (10 mentions)
Matrigel, by BD (9 mentions)
Matrigel, by Corning (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
N acetylcysteine, by Merck Group (5 mentions)
Accutase, by Merck Group (4 mentions)
Y 27632, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
6 well plate, by Thermo Fisher Scientific (3 mentions)
Essential 8 medium, by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Growth factor reduced matrigel, by Corning (3 mentions)
Rock inhibitor, by STEMCELL (3 mentions)
Brain derived neurotrophic factor (bdnf), by R&D Systems (3 mentions)
Polybrene, by Merck Group (3 mentions)
Nicotinamide, by Merck Group (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Human fgf basic, by Thermo Fisher Scientific (3 mentions)
Advanced dmem f12 medium, by Thermo Fisher Scientific (3 mentions)
Ascorbic acid, by Merck Group (3 mentions)
75 protocols using rock inhibitor
1
Murine 2D Colony-Forming Assay
2
Cell Sorting and Flow Cytometry of Transfected iPSCs
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Transfected iPSCs or differentiated cell clusters were dissociated with Accutase (Invitrogen). For sorting, cell suspension was filtered and resuspended in PSB with ROCK inhibitor (Sigma). Fluorescent Activated Cell Sorting (FACS) for Venus-expressing cells was performed on FACS Aria I or II (BD Bioscience). Sorted iPSCs were collected in E8 medium with ROCK inhibitor (Sigma) and seeded at low density to derive single-cell colonies. For flow-based analysis, dissociated cell clusters were fixed for 20 min at 4 °C in cold BD fixation/permeabilizationTM solution (BD Bioscience). Cells were washed twice in BD Perm/WashTM Buffer (BD Bioscience) and incubated with primary antibodies in the dark for 2 hrs at 4 °C. Cells were washed 3x in BD WashTM Buffer (BD Bioscience), resuspended in BD-FBS stainingTM buffer (BD Bioscience) with secondary antibodies and incubated for 1 hr at RT. Primary and secondary antibodies are listed in the Supplementary Data 2 and used at a concentration of 1:100. Stained cells were washed 3x and analysis was performed on FACS Aria II. FlowJo v10 software was used to analyze data. For gating, samples unstained were used as negative controls. Representative flow cytometry pseudocolor plots and gating strategy are shown in Supplementary Fig. 5 .
3
Alveolar Organoid Formation Protocol
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Organoids were produced by combining 5000 AT2 cells with 50,000 fibroblasts (isolated from PND14 wild type C57Bl6 mice and cultured for 3 passages). Cells were mixed in a 1:1 ratio with Matrigel and plated in 24 well plate transwell inserts. Cells were cultured for the first 24 hr with SAGM (Lonza) + Rock inhibitor (Y27632 Sigma), and subsequently cultured in SAGM without Rock inhibitor for 21 days. Organoids were then fixed in 4% PFA overnight, paraffin embedded, and processed for histological analysis. Brightfield images of organoid wells are available in Figure S1 M.
4
Inhibiting Myosin Contraction in Drosophila Wings
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To inhibit myosin contraction during convergent extension, 4 hours APF sqhAX3; {sqh-GFP} wing discs were dissected and transferred, right after peripodial membrane release, to supplemented Shield and Sang medium plus 2.5% methyl-cellulose containing 2.5mM Rock inhibitor (Sigma, Y-27632), and filmed immediately, or alternatively fixed and immunostained after 30 min of inhibitor treatment at 25 C. To inhibit myosin contraction during wing expansion, 5 hours 30 min sqhAX3; {sqh-GFP} wing discs were dissected and transferred to supplemented Shield and Sang medium containing 2.5mM Rock inhibitor, incubated for 3 hours at 25C, fixed and immunostained.
5
Primary UB Isolation and Culture
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The UBtip [23 ] and mK4 cell lines [25 ] were maintained in DMEM/F12 supplemented with 10% FBS, 100 U mL−1 penicillin and 100 µg mL−1 streptomycin and incubated at 37°C in 5% CO2. The cell line tested negative for mycoplasma contamination using the EZ-PCR mycoplasma test kit (Biological Industries). EV-free medium was prepared according to the protocol by Théry et al. [22 ].
The primary UBs were dissected from the kidneys of E11.5 mice embryos (CD-1) by trypsinization. Dissection was performed in EV-free medium supplemented with 10% FBS, 10 ng µl−1 GNDF (RD Systems) and 5 µM ROCK inhibitor (Y-27,632, Sigma) for the first 24 h. After that time, the cells were prepared for vesicle production by cultivation in EV-free medium, DMEM/F12 supplemented with 1% FBS, GDNF and ROCK inhibitor.
The primary UBs were dissected from the kidneys of E11.5 mice embryos (CD-1) by trypsinization. Dissection was performed in EV-free medium supplemented with 10% FBS, 10 ng µl−1 GNDF (RD Systems) and 5 µM ROCK inhibitor (Y-27,632, Sigma) for the first 24 h. After that time, the cells were prepared for vesicle production by cultivation in EV-free medium, DMEM/F12 supplemented with 1% FBS, GDNF and ROCK inhibitor.
6
Generation of iPSCs and Neural Cells
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Generation of iPSCs was performed as described in (Reinhardt et al., 2013 (link)). iPSCs (Table S1 ) where cultured in Matrigel® (Corning, 354277) coated 6‐well plates (Thermo Scientific, 140675), using Essential 8 Basal medium (Thermo Scientific, A1517001) supplemented with ROCK Inhibitor (Y‐27632, Millipore, SCM075) for the first 24 h after plating. The medium was exchanged on a daily basis. Confluence iPSCs (~70%–90%) slipt using Accutase® (Sigma, A6964) and plated at around 300,000 cells per well. Neural progenitor cells needed to generated organoids were derived from iPSCs and maintained in culture as described previously (Nickels et al., 2020 (link); Smits et al., 2019 (link)). iPSCs were also used to generate Macrophage precursors (van Wilgenburg et al., 2013 (link)) and further differentiate them into microglia as described previously (Haenseler, Sansom, et al., 2017 (link)).
7
Directed Differentiation of Hematopoietic Progenitors
8
Neural Differentiation of Human iPSCs
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Human induced pluripotent stem cell (iPSC) colonies were maintained in mTeSR1 medium (STEMCELL Technologies). Neural progenitor cells (NPCs) were derived from iPSCs using STEMdiff Neuron Differentiation Kit (STEMCELL Technologies) and maintained in STEMdiff Neural Progenitor Medium (STEMCELL Technologies). For the neural differentiation, NPCs were plated at 2 × 104 cells/cm2 onto poly-l -ornithine/laminin coated 15-cm dishes in neural differentiation medium [Neurobasal Medium (Gibco), 1× N-2 supplement (Gibco), 1× B-27 supplement (Gibco), 1× GlutaMAX (Gibco), 0.2 μM l -ascorbic acid (Sigma), 1 μM cAMP (Sigma), 10 ng/ml BDNF (Peprotech), 10 ng/ml GDNF (Peprotech)] supplemented with 0.1 μM Compound E (Millipore) and 5 μM ROCK inhibitor (Millipore). Neural cultures were maintained in neural differentiation medium for 1 month before collection. For the iPSC sample collection, cyclohexmide was added to the medium to a final concentration of 100 μg/ml and cells were incubated at 37°C for 1 min. The cells were subsequently washed with ice-cold PBS containing 100 μg/ml CHX, collected by scraping from the dish, pelleted by centrifugation at 15 000g 4°C for 3 min, snap-frozen in liquid nitrogen, and immediately stored at −80°C.
9
Culturing Human Induced Pluripotent Stem Cells
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Human induced pluripotent stem cell culture hiPSCs (CTL07-II iPS) were purchased from the human iPS Core facility at Karolinska Institutet [30]. CTL07-II is a Male cell line, reprogrammed from fibroblasts, with normal 46 XY karyotype. Registration number: 2012/208-31/3.
Coating: 12-well plates were coated with 500 µl laminin-521 (Biolaminina, LN521) diluted 20x in PBS per well at 4℃ overnight or at 37℃ for 2 hours. Medium: hiPSCs were cultured in Complete E8 medium (Gibco, A1517001) with Penicillin-Streptomycin (Sigma, P4333-100ml).
Culturing: hiPSCs were washed once with PBS (Sigma, A6964-500ML). 500 µl TrypleSelect (Gibco, 12563029) were added per well in a 12-well plate for 3 minutes at 37℃. Detached cells were transferred into a tube with 1 ml medium and centrifuged for 3 minutes at 300 g. Afterwards cells were resuspended in fresh medium and 10 µM ROCK Inhibitor (Millipore, Y-27632) was added. 75% of the medium was changed every day and cells were passaged every 3-4 days. Cells were imaged in a ZOE Fluorescent Cell Imager (BioRad).
Coating: 12-well plates were coated with 500 µl laminin-521 (Biolaminina, LN521) diluted 20x in PBS per well at 4℃ overnight or at 37℃ for 2 hours. Medium: hiPSCs were cultured in Complete E8 medium (Gibco, A1517001) with Penicillin-Streptomycin (Sigma, P4333-100ml).
Culturing: hiPSCs were washed once with PBS (Sigma, A6964-500ML). 500 µl TrypleSelect (Gibco, 12563029) were added per well in a 12-well plate for 3 minutes at 37℃. Detached cells were transferred into a tube with 1 ml medium and centrifuged for 3 minutes at 300 g. Afterwards cells were resuspended in fresh medium and 10 µM ROCK Inhibitor (Millipore, Y-27632) was added. 75% of the medium was changed every day and cells were passaged every 3-4 days. Cells were imaged in a ZOE Fluorescent Cell Imager (BioRad).
10
Generation of Healthy Male iPSCs
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The UCSF Committee on human research #10-02521 approved the study protocol for iPSCs. The human iPSC lines used in this study were generated from a healthy male patient, WTC11 (Kreitzer et al., 2013; Miyaoka et al., 2014) (link) using the episomal reprogramming method (Okita et al., 2011) (link). Informed consent was obtained for this procedure. iPSCs were maintained on Matrigel (BD Biosciences) in Essential 8 medium (Life Technologies), which was exchanged every other day. To sparsely populated wells (i.e., passaged wells), we added 10 μM Y-27632, a Rho-associated kinase (ROCK) inhibitor (Millipore), to promote cell survival.
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