To prepare Gli1-expressing cells for RNA sequencing, male mice from different genotypes were injected with TM (1 mg) on P14 or TM (125 μg/g body weight) to activate deletion of AR in Gli1-positive cells. On day E16.5 or P28 or P56, the mice were euthanized and their UGS or prostate tissues were collected, minced into small pieces, and digested with 1 mL of collagenase (10 mg/mL, StemCell Technologies) in DMEM with 10% FBS, DHT (10 nM) and Y-27632 (10 μM) (StemCell Technologies) at 37°C for 90 min. Cells were then digested with TrypLE (1 mL, Gibco) at 37°C for 15 min and were centrifuged at 300xg for 5 min. Cells were passed through 40-μm nylon mesh (Fisherbrand), washed twice with DMEM 10% FBS and dissolved in 500 μL of PBS-2% BSA-1 mg/mL DAPI for cell sorting. Cells were sorted for mGFP-positive and tdTomato-negative, or CD24 positive for prostatic luminal epithelial cells [34 (link)]. After sorting, cells were dissolved in DMEM (100 μL) with 10% FBS and counted using Trypan blue (Gibco). Purity of mGFP-positive cells was confirmed by counting the number of mGFP-positive cells compared to total number of cells which stained negative for Trypan blue. All of the samples used in the study possessed >99% purity.
40 μm nylon mesh
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 40-μm nylon mesh is a laboratory filtration product designed for use in various scientific applications. It features a uniform pore size of 40 micrometers and is constructed from durable nylon material.
Automatically generated - may contain errors
Lab products found in correlation
Tryple, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Collagenase, by STEMCELL (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Y 27632, by STEMCELL (1 mentions)
Haemocytometer, by Nikon (1 mentions)
Wright geimsa stain, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Dispase 2, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
Two step percoll gradient, by GE Healthcare (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Slide angiogenesis, by Ibidi (1 mentions)
Bovine collagen 1, by Thermo Fisher Scientific (1 mentions)
Liver perfusion medium, by Thermo Fisher Scientific (1 mentions)
Liver digest medium, by Thermo Fisher Scientific (1 mentions)
Liberase, by Roche (1 mentions)
Isoflurane, by Pfizer (1 mentions)
9 protocols using 40 μm nylon mesh
1
Isolation and Sorting of Cells for RNA-seq
2
Isolation and Characterization of Murine Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung and liver lobes were cut by scissor into smaller fragments and suspended in media containing tissue digestion enzymes; DNAse I 5mg/mL, Collagenase D 30mg/mL and Dispase II 5mg/mL (all from Roche, Switzerland). Tissue was incubated in digestion buffer in a water bath with shaking at 37°C for 30 minutes. After, liver cells were resuspended in 36% Percoll (Sigma-Aldrich) (adapted from [17 (link)]) and centrifuged for 10mins at 500g without brake to separate leukocytes from fibrous tissue. Spleen was cut into smaller fragments but not subjected to enzymatic digestion. All tissues were strained using a 40μM nylon mesh (Fisher Scientific, USA). Red blood cells were lysed using Ammonium-Chloride-Potassium buffer as previously described [18 (link)]. Cell pellets were resuspended and viable cells counted using Trypan blue staining solution on a haemocytometer (Nikon, Tokyo Japan). Cytospins were performed on glass covered slips stained with Wright-Geimsa stain (Sigma-Aldrich) and imaged using a light microscope (Leica, Germany).
3
Isolation and Purification of Mononuclear Cells from Liver, Lung, and Fat Tissues
4
Bovine Collagen I Angiogenesis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bovine collagen I (600 μl, Life Technologies, Warrington, UK) was mixed with 275 μl water and 100 μl 10× RPMI1640 medium (Thermo Fisher). The pH was neutralised by the addition of 20–25 μl NaOH (until the colour became pink). Colonies were purified using a 40 μm nylon mesh (Fisher Scientific) and mixed with the collagen. Mixes (10 μl) were added to the lower wells of Ibidi angiogenesis slides (Ibidi, Munich, Germany). Antibodies, growth factors or small chemical inhibitors were mixed into the gel at this point (no more than 1:5 vol:vol). Antibodies were added to the gel at 8–10 μg/ml. The gels were allowed to set at 37 °C for 60 min and overlaid with 60 μl medium. Excell 620 medium was replaced every 48 h.
For Matrigel assays, colonies were filtered as above and mixed with a 50:50 mix of Matrigel/Excel medium in Ibidi angiogenesis slides6 .
For Matrigel assays, colonies were filtered as above and mixed with a 50:50 mix of Matrigel/Excel medium in Ibidi angiogenesis slides
5
Isolation of Primary Hepatocytes from GFP-Transgenic Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PHs were isolated from 6 to 11 weeks old GFP-tg. In brief, mice were anesthetized with isoflurane (Pfizer Inc., New York, NY), and the liver was rinsed with 20 ml prewarmed (47 °C) Liver Perfusion Medium (17703-038; Gibco) through the abdominal inferior vena cava. Then, the liver was perfused with Liver Digest Medium (17703-034; Gibco) containing Liberase™-TM (5401127001; Roche). The digested liver was aseptically transferred to sterile tube containing 20 ml cold PBS. Isolated hepatocytes were dispersed into single cell suspension with a large bore pipette, and passed through a sterile 40 μm nylon mesh (Thermo Fisher Scientific Inc.) into a new sterile tube. The cell suspension passed through the mesh was centrifuged at 50×g at 4 °C for 3 min, and the resulting cell pellet was resuspended in cold PBS. Then the cells were counted, and viability of the cells were assessed using the trypan blue exclusion test.
6
Sphere Formation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For analysis of sphere formation, second-generation spheres were thoroughly trypsinized and sieved through a cell strainer with 40-μm nylon mesh (Thermo Fisher) to prepare a single-cell suspension. Cells were suspended in serum-free media (SFM) composed of DMEM/F12 (Gibco), 20 ng/ml EGF, 10 ng/ml bFGF, 10 ng/ml IGF, 2% B27 supplement (Gibco), and 10% Wnt3a-CM, and 200 cells per well were seeded in ultra-low attachment 24-well culture plate (Corning). Varying concentrations of antibodies were added and the cells were fed twice a week by 100 μl SFM. After 14-day incubation, 63 fields in each well were scanned and merged using BIOREVO microscope (BZ-9000; Keyence) to display the whole well image. The number of tumor spheres (>100 μM) was counted or measured from the images. To determine total number of viable sphere-forming cells, spheres were collected by centrifugation and dissociated into single-cell suspension by trypsinization. Cells were washed twice with PBS, stained with 2 μM Calcein-AM for 1 h, and analyzed by flow cytometry.
7
Isolation of Single-Cell Suspension from APA Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh tissue samples from APA patients were rinsed with phosphate-buffered saline (PBS) on ice. Then, each sample was minced and placed into the 30 mL centrifuge tube containing 0.1 mg/mL TrypLE (ThermoFisher Scientific) + 0.1 mg/mL DNAseI (Sigma) in RPMI-1640 (ThermoFisher Scientific) and then incubated for nearly 20 min at 37 °C thermostatic water bath, checking digestion every 10 min in a microscope. Subsequently, 10 mL cold RPMI-1640 containing 10% fetal bovine serum (FBS, ThermoFisher Scientific) was added into the centrifuge tube to terminate the tissues digestion and meantime filtered using 100-μm nylon mesh (ThermoFisher Scientific), transferring to a new 30 mL centrifuge tube. After centrifuging at 300×g for 5 min at 4 °C, the supernatant was discarded and 5 mL 1 × ACK lysis buffer (ThermoFisher Scientific) was added to remove the red blood cells, then filtered again using 40-μm nylon mesh (ThermoFisher Scientific) after the termination of the red blood cells pyrolysis. Finally, the single-cell pellet was resuspended in PBS without calcium and magnesium ions to reach a density of ≤1000 cells/μL. The cell suspension results for different samples are shown in Table S2 .
8
Isolation of Murine Uterine Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse uteri were surgically removed and minced using scissors. Tissues were digested using the MACS Multi Tissue Dissociation Kit II (Miltenyi Biotec) for 80 min at 37 °C. Digested tissues were strained through a 40 μm nylon mesh (ThermoFisher). The Red Cell Lysis Buffer (Miltenyi Biotec) was used to remove red blood cells. Dead cells removed using the MACS Dead Cell Removal Kit (Miltenyi Biotec), and EPCAM-positive cells were positively selected and purified using a PE-conjugated EPCAM antibody and anti-PE MicroBeads (Miltenyi Biotec), per the manufacturers’ instructions. A BD Accuri C6 flow cytometer (BD Biosciences) was used to confirm purity of EPCAM-positive population.
9
Evaluating Cell Cycle Kinetics in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C57BL/6 mice were administered daily oral gavages of 10, 50 or 100 mg/kg of G1T38 or palbociclib for 7 consecutive days. Eleven hours later, each mouse was given a single IP injection of 10 mg/kg EdU and femurs harvested 1 hours later (3 mice/dose/time-point). All protocols were IACUC approved and experiments were completed at Integrated Laboratory Systems. Bone marrow cells were isolated by flushing the femurs using ice-cold staining medium (1× Ca2+- and Mg2+-free Hank's balanced salt solution (HBSS, Gibco) supplemented with 10 mM EDTA (Corning) and 2% heat-inactivated bovine serum (Gibco)) and filtered through 40-μm nylon mesh (ThermoFisher). The cells were then fixed in 5% formalin for 10 minutes on ice and permeabilized in staining medium plus 0.1% saponin for 10 minutes at room temperature. EdU staining was completed using manufactures’ instructions (Life Technologies). Flow cytometry analysis of stained cells was completed on a LSR II 3-laser flow cytometer (Becton-Dickinson), and recorded data was analyzed in FlowJo 10.0.8 software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!