Astrocytes were nucleofected at DIV 7 using the Basic Nucleofector Kit for Primary Mammalian Glial Cells (#VPI-1006; Lonza) according to the manufacturer’s protocol. Briefly, astrocytes were trypsinized and cell density was determined. For each condition, the appropriate volume for 2 million cells was added to 15-ml tubes, centrifuged at 11 min at 200 g, and the resulting supernatant was removed completely. The cell pellet was resuspended in 100 μl of room temperature Basic Nucleofector Solution for Mammalian Glial cells and 5 μg of shRNA plasmid. The resulting cell/plasmid suspension was immediately transferred into a cuvette and nucleofected using the T-20 program for Nucleofector 2b Device. After nucleofection, 500 μl of pre-equilibrated NGM Plus was added to the cell/plasmid suspension. Cell density of live cells after nucleofection was determined and nucleofected astrocytes were plated at a density of 80,000/coverslip (for neuron morphology) or 300,000/well of a 6-well plate (for astrocyte surface biotinylation assay). For astrocyte mosaic preps, nucleofected astrocytes were first cultured in AGM for 24 h before being switched to NGM Plus for another 48 h.
Basic nucleofector kit for primary mammalian glial cells
Manufactured by Lonza
Sourced in United States
The Basic Nucleofector Kit for Primary Mammalian Glial Cells is a laboratory equipment product designed for the transfection of primary glial cells. It provides the necessary solutions and procedures for efficient gene transfer into these cell types.
Automatically generated - may contain errors
5 protocols using basic nucleofector kit for primary mammalian glial cells
1
Astrocyte Nucleofection for Morphology and Surface Biotinylation
2
Silencing AMPK Subunits in Oligodendrocyte Progenitors
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Nucleofection of isolated OPCs was performed with siRNA mixtures to target both AMPKα1 (Dharmacon, Cat. No. L-091373-02-0005) and AMPKα2 (Dharmacon, Cat. No. L-100623-02-0005), using the basic nucleofector kit for primary mammalian glial cells (Lonza Biosciences). Control cells were transfected with a pool of non-targeting siRNAs (Dharmacon). OPCs were grown in high growth factor media (1x SATO with 20 ng/mL PDGFRα and 20 ng/mL FGF) at 37°C + 7.5% CO2 for 24 h prior to transfection. After trypsinization, cells were spun down and resuspended in the nucleofector resuspension solution according to the manufacturer’s protocol. Cells were then returned to the 37°C + 7.5% CO2 incubator for 1 to 1.5 h prior to siRNA transfection with 8 μL of AMPKα1 siRNA at a stock concentration of 20 μM and 6 μL of AMPKα2 siRNA at a stock concentration of 20 μM. Post-transfection, cells were immediately resuspended in the high growth factor media and plated in tissue culture treated petri dishes for 24 h after which the media was changed to OL differentiation media (1x SATO + T3).
3
Efficient siRNA Knockdown in OPCs
4
Silencing Lrp1 in Primary Microglia
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Mouse Lrp1-specific siRNAs and non-targeting control were purchased from Dharmacon Research. The siRNA sequences for mouse Lrp1 were as followed: Lrp1 siRNA1: 5′-GGAGUCACUUACAUCAAUAUU-3′; Lrp1 siRNA2: 5′-GCAGCGAGCCAACAAG UAU-3′. siRNAs were transfected into primary microglia using Basic Nucleofector™ Kit for Primary Mammalian Glial Cells (VPI-1006, Lonza) according to the manufacturer’s specifications. Each electroporation reaction contained 6 × 106 cells and 300 nM siRNA. Cells were further cultured for 48 h before using in experiments and analysis.
5
CALR Knockdown in Glioblastoma Cells
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Human CALR siRNA and Universal Scrambled Negative control siRNA were purchased from Origene (Cat#SR300567, Origene, Rockville, MD, USA). Transfection was by Nuclefection (Amaxa Biosystems Inc., Gaithersburg, MD, USA). Basic nucleofector kit for primary Mammalian Glial cells (Cat#VPI-1006, Lonza, Allendale, NJ, USA) was purchased and optimized for nucleofection of GBM cells according to manufacturer’s recommendation. Then, 2–3 × 106 cells were suspended in 100 μL Basic NucleofectorTM solution and mixed with 300 nM (30 pmol/sample) CALR siRNA or Universal scrambled negative control siRNA. The cell-siRNA mixtures were then transferred to a Lonza-certified cuvette and subjected to nucleofection using a pre-existing program (T-020) on the nucleofector device. Immediately after nucleofection, 500 μL of the culture medium was added to cells in the cuvette, and cells were transferred to a culture dish containing complete growth medium and incubated in a humidified CO2 incubator at 37 °C. Transfection efficiency was assessed using pMax-GFP. CALR-knockdown was confirmed by immunoblot analysis.
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