Primary human cortical astrocytes (ScienCell) were obtained commercially and expanded in astrocyte media (ScienCell). Cells were cultured at 37°C in 5% CO2 and split at approximately 80% confluency until passage 3. Upon reaching passage 3, cells were frozen using Recovery Cell Freezing Media (Gibco). Cells were thawed prior to experiments and transfected during passage 4. Vectors encoding either intron 2 retained PSMB8 (i2R-PSMB8) or the canonical full-length transcript (FL-PSMB8) in pcDNA3.1 were obtained from GenScript, outgrown in DH5α E. coli (New England Biolabs), and midi-prepped using a Plasmid Plus Midi kit (Qiagen) per manufacturer’s instructions. Vector maps are provided as Supplementary Figures S1, S2 . Vectors were transfected using a Lonza Amaxa Nucleofector 4 device, with kit P3 and protocol DR114, at a ratio of 6 μg DNA per 106 cells. This resulted in a 50–55% transfection efficiency (Supplementary Figure S3 ).
Recovery cell freezing media
Manufactured by Thermo Fisher Scientific
Recovery Cell Freezing Media is a sterile, ready-to-use cryopreservation solution designed to protect cells during freezing and storage. The media contains a balanced salt solution, cryoprotectants, and stabilizers to help preserve cell viability and functionality upon thawing.
Automatically generated - may contain errors
2 protocols using recovery cell freezing media
1
Transfection of Astrocytes with PSMB8 Variants
2
Transfection of Primary Human Astrocytes
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Primary human cortical astrocytes (ScienCell) were obtained commercially and expanded in astrocyte media (ScienCell). Cells were cultured at 37°C in 5% CO2 and split at approximately 80% confluency until passage 3. Upon reaching passage 3, cells were frozen using Recovery Cell Freezing Media (Gibco). Cells were thawed prior to experiments and transfected during passage 4. Vectors encoding either intron 2 retained PSMB8 (i2R-PSMB8) or the canonical full-length transcript (FL-PSMB8) in pcDNA3.1 were obtained from GenScript, outgrown in DH5α E. coli (New England Biolabs), and midi-prepped using a Plasmid Plus Midi kit (Qiagen) per manufacturer’s instructions. Vector maps are provided as Supplemental Figures 1 –2 . Vectors were transfected using a Lonza Amaxa Nucleofector 4 device, with kit P3 and protocol DR114, at a ratio of 6 μg DNA per 106 cells. This resulted in a 50–55% transfection efficiency (Supplemental Figure 3 ).
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