Astrocytes were isolated from 4.5-mo-old wild-type and rTg4510 (n = 9) mice as described previously (Swartzlander et al., 2018 (link)). In brief, mice were anesthetized and perfused with ice-cold saline. The forebrain was dissected and then finely minced using a razor blade. The minced forebrain was resuspended in HBSS and enzymatically digested in a solution containing papain (LK003178; Worthington Biochemical Corporation) and DNase. The suspension was then triturated with a glass Pasteur pipette to mechanically dissociate the minced forebrain. Cellular debris and myelin were removed by filtration through a 40-µm filter and centrifugation in 21% isotonic percoll resulting in a pellet of single cells. To isolate astrocytes from the dissociated singles cells, the cells were first blocked in HBSS with 0.5% BSA and mouse Fc Block (BD Biosciences) before staining for viability with LIVE/DEAD blue (L23105; Thermo Fisher) and with the fluorescent antibodies CD45 and ACSA-2. Approximately 200,000 live ACSA-2 positive, CD45 negative astrocytes were isolated per brain through FACS using a BD FACSAria II.
Lk003178
Manufactured by Worthington
The LK003178 is a laboratory equipment item designed for general laboratory use. It serves a core function of providing a specific capability required in laboratory settings. No further details about its intended use or functionality are provided.
Automatically generated - may contain errors
Lab products found in correlation
Papain, by Worthington (2 mentions)
Live dead blue, by Thermo Fisher Scientific (1 mentions)
Mouse fc block, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
P4707, by Merck Group (1 mentions)
Lk003172, by Worthington (1 mentions)
Lk003182, by Worthington (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
2 protocols using lk003178
1
Isolation of Astrocytes from Mouse Brains
2
Primary Culture of Cortical Neurons and Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mixed cultures of cortical neurons and astrocytes were obtained by dissecting brains from P0-P1 mice. Cortices from each brain were isolated, kept in HBSS (H6648, Sigma) on ice separated from each other and genotyping was performed on liver extracted from each pup.
Brain tissue was incubated in EBSS (E2888, Sigma) and papain (LK003178, Worthington Biochemical Corp.) for 40 min at 37 °C and it was then dissociated by trituration in EBSS supplemented with DNAse (LK003172, Worthington Biochemical Corp.) and papain inhibitor (LK003182, Worthington Biochemical Corp.). After spinning, the cell pellet was resuspended in Neurobasal (21103-049, Life Technologies), supplemented with B27 (17504-044, Life Technologies), Glutamax (35050-038, Life Technologies), and 100 U/ml Penicillin–Streptomycin (1514-122, Life Technologies) counted and plated to appropriate densities on coverslips (0.5·106 cells), 6-well plates (106 cells) or 96-well plates (30000 cells), coated with polylysine (P4707, Sigma). Half media changes were done every 4 or 7 days. Cultures were maintained at 37 °C and 5% CO2 in humidified atmosphere and used between 10 and 15 days in vitro unless stated differently.
Brain tissue was incubated in EBSS (E2888, Sigma) and papain (LK003178, Worthington Biochemical Corp.) for 40 min at 37 °C and it was then dissociated by trituration in EBSS supplemented with DNAse (LK003172, Worthington Biochemical Corp.) and papain inhibitor (LK003182, Worthington Biochemical Corp.). After spinning, the cell pellet was resuspended in Neurobasal (21103-049, Life Technologies), supplemented with B27 (17504-044, Life Technologies), Glutamax (35050-038, Life Technologies), and 100 U/ml Penicillin–Streptomycin (1514-122, Life Technologies) counted and plated to appropriate densities on coverslips (0.5·106 cells), 6-well plates (106 cells) or 96-well plates (30000 cells), coated with polylysine (P4707, Sigma). Half media changes were done every 4 or 7 days. Cultures were maintained at 37 °C and 5% CO2 in humidified atmosphere and used between 10 and 15 days in vitro unless stated differently.
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!