For transfecting primary neurons with siRNA, are there any recommended transfection reagents with low cytotoxicity?

Primary neurons are sensitive to liposomes. It is recommended to first reduce the final siRNA concentration from 50 nM to 20-30 nM. Many neuronal target genes can be knocked down at 20 nM, and toxicity will be considerably lower. For the reagent, you can try Lipofectamine RNAiMAX, using 0.5-1 μL per well in a 24-well plate, and replace half of the medium 4 h after transfection. A 0.1 mL small packaging is available; if funding is limited, you can apply for a trial sample. The efficiency is not as high as in tumor cells, but neuronal morphology and survival rate are better than with regular Lipofectamine 2000.

If funding is limited and you want a gentle option, consider non-liposomal transfection reagents, such as Mirus TransIT-siQUEST or Qiagen HiPerFect. Their toxic side effects are usually lower, and some can be used for transfection in full medium without dedicated Opti-MEM. In a 24-well plate at 50 nM, use HiPerFect at 3 μL/well, and do not change the medium after transfection. I have done this with primary hippocampal neurons, and the mortality rate was controllable. First contact a regional distributor to request a 0.1/0.25 mL trial sample, and use β-actin or GAPDH siRNA to verify knockdown before adding the target gene, to avoid waste.

For primary neurons, I recommend optimizing both the "low-toxicity reagent" and the "medium change time after transfection" together, rather than relying solely on the brand. A lot of the cell death is caused by prolonged exposure to the transfection complex. You can try a half-volume medium change with complete medium at 1.5-2 h after transfection, and use 50 nM siRNA per well while reducing the reagent to the minimum amount specified in the manual. In terms of brands, Thermo's RNAiMAX, Mirus's TransIT-siQUEST, and Biontex K2 all have primary neuron cases. First request trial samples, and validate with both a cell viability kit and qPCR at the same time, not just transfection fluorescence.

Primary neurons are really picky about reagents. It is recommended to first test toxicity in less critical wells, and the siRNA concentration should also be optimized using a gradient.

Same here. I previously used a reagent for primary culture, and the cell morphology changed, so I don't dare try it again.