Entosis was assayed as previously described (Sun and Overholtzer, 2013 (link)). Briefly, cells were trypsinized, washed, and resuspended in complete medium at 4 × 105 cells per milliliter. For entosis after 6 h, 0.5 ml was plated in a well of a Costar 24-well Ultra-Low attachment plate (Corning; 3473). After 6-h incubation, cells were centrifuged and thoroughly resuspended in 400 µl PBS, and then 200 µl was Cytospun onto a glass slide for 5 min at 500 rpm. Cells on slides were fixed in 2% formaldehyde (diluted from 16% methanol-free formaldehyde; Thermo Fisher Scientific; 28908), and then mounted with a coverslip in PBS. For the 0-h time point of entosis, cells were processed for cytospin as above immediately after trypsinization. High-power 20× fields (comprising ∼300 to 1,000 total cells each) were captured on a phase-contrast Olympus microscope (below). Cells undergoing entosis (a positive was scored as any cell with one half or more of another cell body internalized inside of it) and total cells were scored as previously described (Sun and Overholtzer, 2013 (link)) and are shown as individual data points. ROCK inhibitor Y-27632 (10 µM; Apex Bio) was added to cells at the time of plating in low-adhesion plates.
Rock inhibitor y 27632
Manufactured by Apexbio
ROCK inhibitor Y-27632 is a small molecule that inhibits the activity of Rho-associated protein kinase (ROCK) enzymes. ROCK inhibitors can be used in research applications to study the role of ROCK signaling in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Costar 24 well ultra low attachment plate, by Corning (1 mentions)
Aria fusion, by BD (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Dispase 2, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase hyaluronidase, by STEMCELL (1 mentions)
R spondin 1, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Wnt7b, by Abcam (1 mentions)
Gts 21, by Abcam (1 mentions)
Collagen coated transwell inserts, by Corning (1 mentions)
Cftr inhibitor 172, by Merck Group (1 mentions)
Pf 04418948, by Cayman Chemical (1 mentions)
L 161 982, by Cayman Chemical (1 mentions)
Pneumacult ali, by STEMCELL (1 mentions)
4 protocols using rock inhibitor y 27632
1
Quantifying Entosis in Cell Cultures
2
Prostate Cell Isolation and Phenotyping
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Whole prostate tissue from a minimum of three mice were dissected and pooled in cold PBS 2% FBS, minced and digested at 37°C for 2 hr in DMEM 5% FBS 1X Collagenase/hyaluronidase (Stemcell Technologies) for 5 min in 0.25% trypsin/EDTA and followed by 10 min in dispase II (5 U/ml) and DNase I (0.1 mg/ml) (Roche). All solution contained 10 μM of Rock inhibitor Y-27632 (ApexBio). The digested cells were passed through a 27G needle and filtered through a 70 μm cell strainer. Cell staining was done on ice for 30 min using human IgG blocking solution and antibodies CD45 (30-F11), TER119 (TER-119), CD31 (MEC13), CD49f (GoH3), EpCAM (G8.8), SCA1 (D7) (all from Biolegend) and TROP2 (from R and D). Dead cells were excluded by fixable Viability dye (eBioscience) staining. FACS analysis and sorting was performed on a BD Fortessa and Aria Fusion apparatus (BD Biosciences). Data was analyzed using DIVA (BD Biosciences) and FlowJo softwares.
3
Establishing Lung Organoids from Lineage-Labeled Cells
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Lung organoids were established based on a previous report with modifications (48 (link)). Briefly, freshly sorted lineage-labeled cells (CD45–CD31–EPCAM+tdTomato+) were resuspended in basic media (advanced DMEM/F12 supplemented with 10% FBS and mixed with lung mesenchymal cells (CD45–CD31–EPCAM–) at a ratio of 1:6, followed by resuspension in growth factor–reduced Matrigel (BD Biosciences) at a ratio of 1:5. A 10 μL mixture was placed in a prewarmed 24-well Transwell insert with a 0.4 mm pore (Corning) or 48-well cell culture plate at 37°C for 20 minutes. Approximately 2 × 103 to 5 × 103 Sftpc+ cells were seeded in each insert. 500 μL or 250 μL organoid media (listed in Supplemental Table 2 ) was placed in the lower chamber, and medium was changed every 3 days with or without LPS (1 μg/mL, Sigma-Aldrich), GTS-21 (10 μmol/L, Abcam), MLA (10 μmol/L, Abcam), WNT7B (100 ng/mL, referred to in refs. 49 (link), 50 (link); Novus Biologicals). R-Spondin-1 (500 ng/mL, Peprotech) and ROCK inhibitor Y-27632 (5 μmol/L, APExBio) were added in the medium for the first 3 days of culture. Colony-forming efficiency and size of organoids were analyzed at day 10 after plating if there is no specific description.
4
Isolation and Culture of Human Airway Epithelial Cells
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Human tracheal epithelial cells were harvested from deceased organ donors according to established protocols (70 (link)). Human sinus epithelial cells were harvested from research participants undergoing endoscopic surgery using a cytobrush as described above. For Ussing chamber measurements, the inferior turbinate of healthy individuals was sampled using a protocol approved by the National Jewish Health Institutional Review Board (HS-2832), and all donors provided written informed consent prior to the procedure. Tracheal, sinus, or nasal epithelial cells were seeded onto mitomycin-treated MRC5 or irradiated NIH 3T3 fibroblast feeder layers (both from ATCC) and cultured in cultured in conditional reprogramming culture medium (71 (link)) supplemented with the ROCK inhibitor Y-27632 (ApexBIO). Expanded cells were plated on collagen-coated Transwell inserts (Corning) cultured with Pneumacult ALI (StemCell Technologies) for 21–28 days according to the manufacturer’s instructions.
For organoid culture, expanded basal cells were plated in matrix (80% Matrigel, 20% media) in Pneumacult Airway Organoid Media (StemCell Technologies) according to manufacturer’s instructions for 21–24 days. Cells were stimulated with PGE2 (Sigma, 1 μg/mL) daily, EP2 inhibitor (PF-04418948, Cayman Chemical, 10 μM), EP4 inhibitor (L-161,982, Cayman Chemical, 10 μM), or CFTR inhibitor 172 (Sigma, 10 μM).
For organoid culture, expanded basal cells were plated in matrix (80% Matrigel, 20% media) in Pneumacult Airway Organoid Media (StemCell Technologies) according to manufacturer’s instructions for 21–24 days. Cells were stimulated with PGE2 (Sigma, 1 μg/mL) daily, EP2 inhibitor (PF-04418948, Cayman Chemical, 10 μM), EP4 inhibitor (L-161,982, Cayman Chemical, 10 μM), or CFTR inhibitor 172 (Sigma, 10 μM).
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