For immunofluorescence staining, the colonoids were collected from Matrigel with Cell Recovery Solution (Corning, #354253) and fixed with 4% PFA for 30 min. The next steps were the same as performed in the tissue sections (described above). The BeyoClick™ EDU Cell Proliferation Kit with Alexa Fluor 594 (C0078S, Beyotime Biotechnology, Shanghai, China) was used to evaluate cell proliferation in colonoids. Briefly, the colonoids were treated with EDU (10 μM) at 37 °C for 2 h and then fixed in 4% PFA at room temperature for 15 min. After permeabilization with 0.3% Triton for 15 min, the colonoids were reacted with reaction mixture fluid for 30 min. The nuclei were stained with DAPI for 10 min. The images of the colonoids were captured using a fluorescence microscope (Axio Observer 7, Carl Zeiss). The ratio of positive cells was calculated as previously described.
Matrigel with cell recovery solution
Manufactured by Corning
Matrigel with Cell Recovery Solution is a cell culture matrix product. It provides a reconstituted basement membrane extract that supports the attachment and growth of various cell types in vitro. The Cell Recovery Solution is used to safely and efficiently recover cells from the Matrigel matrix.
Automatically generated - may contain errors
Lab products found in correlation
Beyoclick edu cell proliferation kit with alexa fluor 594, by Beyotime (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Dispase, by Corning (1 mentions)
Dispase, by BD (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Epcam pe cy7, by BioLegend (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Y 27632, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
2 protocols using matrigel with cell recovery solution
1
Immunofluorescence Analysis of Colonoid Proliferation
2
LGR5+ Intestinal Stem Cell Isolation
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Isolated crypts from GFP-LGR5 mice were incubated in HBSS without Ca2+ and Mg2+ supplemented with 0.3 U/mL Dispase (Corning), 0.8 U/μL DNase (Sigma), and 10 μM Y-27632 (Sigma) for 30 min at 37 °C. For cell sorting, dissociated cells were washed with 1% BSA/PBS and stained with CD24-APC antibody (1:20; BioLegend; M1/69) and EpCam Pe-Cy7 (1:100; BioLegend; G8.8) for 20 min at 4 °C. The cells were then resuspended in crypt media supplemented with 10 μM Y-27632, filtered with a 35-μm mesh, and analyzed by MoFlo Astrios (Beckman Coulter). Sorted cells were collected in RLT buffer (Qiagen). For flow cytometric analysis, GFP-LGR5 or ATG16L1 KO organoids were removed from the Matrigel with Cell Recovery Solution (Corning), dissociated with Dispase solution (as for the isolated crypts), stained, and acquired on a FACSCanto II (BD Biosciences) or an Attune NxT flow cytometer (Thermo Fisher) and analyzed by FlowJo software (Tree Star). GFP-LGR5 ISC concentration was measured using CountBright absolute counting beads (Thermo Fisher).
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