The uterine tissues from 3 mice for each group were pooled and minced with a blade. Tissues were then incubated in the dissociation buffer containing 2 mg/ml Collagenase II (#C6885, Sigma‐Aldrich), 10 mg/ml Dispase II (#354235, Corning) and 50,000 U/ml DNase I (#DN25, Sigma‐Aldrich) for up to 30 min at 37°C in a shaking incubator. The digestion progress was checked every 5 min with a microscope until single‐cell suspension was achieved. The single‐cell suspension was then passed through a 40‐μm cell strainer to remove undigested tissues. Cells were spun down at 250 g at 4°C for 4 min, and the pelleted cells were washed using centrifugation. In order to measure cell viability, cells were stained with AO/PI solution (#CS2‐0106, Nexcelom Bioscience) and counted using a Cellometer Auto 2000 instrument (#SD‐100, Nexcelom Bioscience). The single‐cell suspension was carried forward to single‐cell RNA‐seq only if the cell viability was >80% and the percentage of cell clumps was <10%.
Dispase 2
Manufactured by Corning
Sourced in United States
Dispase II is a non-specific neutral protease derived from Bacillus polymyxa. It is capable of dissociating a variety of cell and tissue types, including epithelial, endothelial, and connective tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (6 mentions)
Collagenase 2, by Merck Group (5 mentions)
Ao pi solution, by Revvity (5 mentions)
Cellometer auto 2000 instrument, by Revvity (4 mentions)
Dnase, by Thermo Fisher Scientific (3 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Cd16 cd32, by BD (1 mentions)
F12 media, by Thermo Fisher Scientific (1 mentions)
Its supplement, by Merck Group (1 mentions)
Hiseq sequencing, by Illumina (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Dispase 2, by Merck Group (1 mentions)
Low melting point agarose, by Sangon (1 mentions)
Streptavidin microbeads, by Miltenyi Biotec (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using dispase 2
1
Dissociation and Viability Assay for Uterine Cells
2
Lung Tissue Dissociation for Bacterial Burden Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were euthanized with a lethal dose of sodium pentobarbital (Socumb 6G, 150 mg/kg). Lungs were inflated via tracheal cannulation with 1 ml digestion buffer: RPMI (Gibco) with 2 mg/ml collagenase IV (Gibco), 3 mM CaCl2, 5 U/ml DNase (Thermo Scientific), 5 U/ml dispase II (Corning), 5% fetal bovine serum (Sigma), and 10 mM HEPES buffer (Affymetrix). Lungs were excised from the mouse, placed in 5 ml digestion buffer, and incubated for 45 min at 37°C with 5% CO2 with periodic agitation. Room temperature PBS was then added to each digestion to bring the volume to 30 ml, and samples were vortexed to create a single-cell suspension. For determination of bacterial burden, a portion of this lung cell suspension was removed, and serial dilutions were plated on BHI agar (for Y. pestis ) or LB agar with 30 μg/ml rifampin (for K. pneumoniae ) and reported as CFU/lung. The remaining cell suspension was filtered through a 70-μm filter and centrifuged at 700 × g for 5 min at 4°C. The cell pellet was resuspended in 2 ml ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) for 2 min to lyse erythrocytes. Eighteen milliliters of ice-cold flow buffer (PBS with 5% fetal bovine serum [FBS], 2 mM EDTA, 0.1% sodium azide) was added to each sample, and samples were filtered through 70-μm filters. Lung single-cell suspensions were kept on ice until antibody staining.
3
Isolation and Genetic Manipulation of Primary Pneumocytes
4
Isolation of Mouse Alveolar Type II Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adult mouse lungs were used for the isolation of AT2 cells [11 (link)]. In brief, the lung was perfused with 4 °C PBS. After trachea intubation, dispase II (3 mg/mL, Corning, New York, NY, USA) was injected into the lung, followed by 1% low-melting-point agarose (42–45 °C, Sangon Biotech, Shanghai, China). Then, the lung was incubated in dispase II for 45 min at 37 °C and gently torn in DMEM/F12 + DNase I (0.01%, Sigma, St. Louis, MO, USA). The cell suspension was passed through serial filters (Solarbio, Beijing, China) and incubated with biotinylated antibodies: CD16/32 and CD45 (Miltenyi Biotec, Shanghai, China) for 10 min. After being incubated with 10 µL Streptavidin MicroBeads (4 °C, Miltenyi Biotec, Shanghai, China) for 15 min in dark, the cells were transferred onto plates pre-coated with 1 mg/mL IgG for 2 h, and the unattached cells were collected. The cell purity was detected by flow cytometry with corresponding antibodies.
5
Single-cell Isolation from Murine Uterus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Uterine samples from 3 mice for each group were pooled and minced with a blade. Samples were then incubated in dissociation buffer containing 2 mg/mL Collagenase II (#C6885, Sigma-Aldrich, St. Louis, MO, USA), 10 mg/mL Dispase II (#354235, Corning, Corning, NY, USA) and 50,000 U/mL DNase I (#DN25, Sigma-Aldrich, St. Louis, MO, USA) for up to 30 min at 37 °C in a shaking incubator. The digestion progress was checked every 5 min with a microscope until a single cell suspension was achieved. The single-cell suspension was then passed through a 40-micrometer cell strainer to remove undigested tissues. Cells were spun down at 250× g at 4 °C for 4 min and the pelleted cells were washed using centrifugation. In order to measure cell viability, cells were strained with AO/PI solution (#CS2-0106, Nexcelom Bioscience, Lawrence, MA, USA) and counted using a Cellometer Auto 2000 instrument (#SD-100, Nexcelom Bioscience, Lawrence, MA, USA). The single-cell suspension was carried forward to single-cell RNA-seq only if the cell viability was >80% and the percentage of cell clumps was <10%.
6
Isolation of Single-Cell Suspension from Murine Uterine Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspension was prepared as described previously [16 , 17 ]. Briefly, the uterine tissues from 3 mice for each group were pooled and minced with a blade. Tissues were then incubated in dissociation buffer containing 2 mg/ml Collagenase II (#C6885, Sigma-Aldrich), 10 mg/ml Dispase II (#354,235, Corning) and 50,000 U/ml DNase I (#DN25, Sigma-Aldrich) for up to 30 min at 37 °C in a shaking incubator. The digestion progress was checked every 5 min with a microscope until a single cell suspension was achieved. The single-cell suspension was then passed through a 40-μm cell strainer to remove undigested tissues. Cells were spun down at 250 g at 4 °C for 4 min and the pelleted cells were washed using centrifugation. In order to measure cell viability, cells were strained with AO/PI solution (#CS2-0106, Nexcelom Bioscience) and counted using a Cellometer Auto 2000 instrument (#SD-100, Nexcelom Bioscience). The single-cell suspension was carried forward to single-cell RNA-seq only if the cell viability was > 80% and the percentage of cell clumps was < 10%.
7
Uterus Single-Cell Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole uterus segments were incubated in the dissociation buffer containing 2 mg/mL Collagenase II (#C6885, Sigma-Aldrich, St. Louis, MO, USA), 10 mg/mL Dispase II (#354235, Corning, Corning, NY, USA) and 50,000 U/mL DNase I (#DN25, Sigma-Aldrich, St. Louis, MO, USA) for up to 30 min at 37 °C in a shaking incubator. The digestion progress was checked every 5 min with a microscope until single-cell suspension was achieved. The single-cell suspension was then passed through a 40-μm cell strainer to remove undigested tissues. Cells were spun down at 250× g at 4 °C for 4 min and the pelleted cells were washed using centrifugation. In order to measure cell viability, cells were strained with AO/PI solution (#CS2-0106, Nexcelom Bioscience, Lawrence, MA, USA) and counted using a Cellometer Auto 2000 instrument (#SD-100, Nexcelom Bioscience, Lawrence, MA, USA). The single-cell suspension was carried forward to single-cell RNA-seq only if the cell viability was >80% and the percentage of cell clumps was <10%.
8
Porcine Endometrial Single-cell Dissociation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell dissociation was performed as described previously (Yang et al., 2021a (link); He et al., 2021 (link)). Endometrial tissues and conceptus fragments from 3 gilts for each group were minced with a blade and then incubated in dissociation buffer containing 2 mg/ml Collagenase II (#C6885, Sigma-Aldrich), 10 mg/ml Dispase II (#354235, Corning) and 50,000 U/ml DNase I (#DN25, Sigma-Aldrich) for up to 30 min at 37°C in a shaking incubator. The digestion progress was monitored with a microscope until a single cell suspension was achieved. To remove undigested tissues, the single-cell suspension was then passed through a 40-μm cell strainer and cells were spun down at 250 g at 4°C for 4 min. Red blood cells (RBC) were removed by using RBC Lysis Buffer (#00-4333, Invitrogen). Cell viability was measured by AO/PI solution (#CS2-0106, Nexcelom Bioscience). The quality control criteria for single-cell suspension were cell viability > 80% and the percentage of cell clumps < 10%.
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!