iPSC colonies were dissociated into a single-cell suspension using Accutase (Thermo Fisher, A11105-01) and resuspended in E8 medium containing 10 μM Rock inhibitor. Cells were counted using an automated cell counter (Chemometec NC-200) and a cell suspension containing an equal amount of each iPSC line was prepared in E8 medium containing 10 μM Rock inhibitor and seeded at 2 × 105 cells per cm2 on 1% Geltrex- (Thermo Fisher, A1413202) coated plates. Each pool of lines contained between 7 to 24 donors. 24 h after plating, neuronal differentiation of the pooled lines to a midbrain lineage was performed as described by13 (link) with minor modifications: 1. SHH C25II was replaced by 100 nM SAG (Tocris, 6390) in the neuronal induction phase. 2. On day 20, the cells were passaged with Accutase containing 20 units/ml of papain (Worthington, LK00031765) and plated at 3.5 × 105 cells per cm2 on 1% Geltrex-coated plates for final maturation. A link to the step-by-step protocol can be found in the URL section .
Accutase
Manufactured by Worthington
Accutase is a cell detachment solution that is used for the dissociation of adherent cells. It is a mixture of enzymes that can effectively break down the extracellular matrix, allowing for the gentle and efficient harvesting of cells from culture.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (4 mentions)
Collagenase 4, by Worthington (4 mentions)
Dnase type 4, by Merck Group (3 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Nc 200, by ChemoMetec (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Papain, by Worthington (1 mentions)
Deoxyribonuclease type 4, by Merck Group (1 mentions)
5 protocols using accutase
1
iPSC-Derived Midbrain Neuron Protocol
2
Tumor Dissociation for Single-Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors were mechanically digested with accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma), and deoxy-ribonuclease type IV (Sigma). Single-cell suspensions were then prepared and stained for flow cytometry analysis (FACS) or CD45-positive cell enrichment.
3
Non-Small Cell Lung Cancer Tumor Sampling and Processing
4
Non-Small Cell Lung Cancer Tumor Sampling and Processing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh tumor samples were collected from individuals with non-small cell lung cancer undergoing primary surgical treatment between January 2013 and June 2016 at the University Hospital Basel, Switzerland and at the Ortenau Klinikum, Germany. Paraffin-embedded tumor biopsies were collected from stage IV non-small cell lung cancer patients undergoing treatment with nivolumab between April 2015 and June 2017 at the University Hospital Basel and the Cantonal Hospital Baselland, Switzerland. Detailed patient characteristics are provided in Supplementary Table 3 and 4 . The study was approved by the local Ethical Review Board (Ethikkommission Nordwestschweiz), performed in compliance with all relevant ethical regulations, and all patients consented in writing to the analysis of their tumor samples.
Resected solid tumor lesions were immediately processed into single cell suspensions by mechanical dissociation and enzymatic digestion using accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma) and DNAse type IV (Sigma). All samples were cryopreserved until further usage.
Resected solid tumor lesions were immediately processed into single cell suspensions by mechanical dissociation and enzymatic digestion using accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma) and DNAse type IV (Sigma). All samples were cryopreserved until further usage.
5
Immune Cell Isolation from MC38 Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For analysis of immune populations isolated from MC38 tumors, the following protocol was used. Harvested MC38 tumors were cut into smaller fragments with razor blades and then placed in 2–4 mL digestion mix [containing accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma), and DNAse type IV (Sigma)] at 37°C for 45 min with constant shaking. Tumor suspensions were then filtered via 70 μM nylon mesh and washed with PBS containing EDTA (5 mM). Cell suspensions were subjected to red blood cell (RBC) lysis by adding 1 mL of RBC lysis buffer for 1 min, followed by a wash with PBS containing 2 mM EDTA. As a final step, cell suspensions were filtered via 70 μM nylon mesh and either stored in −80°C until further analysis or used directly for flow cytometry staining.
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!