Calcium phosphate transfection kit

Two different lentiviruses containing human Neurogenin2 (hNGN2) under the control of TetON promoter or reverse tetracycline activator (rtTA) were generated. Each lentivirus was produced by co‐transfecting the transgene construct with two helper plasmids, pMD2 and psPAX2, into HEK293 cells using calcium phosphate transfection kit (Takara) following the manufacturer's protocol. After 48–72 h of incubation, media containing lentiviruses were harvested and concentrated at 28,000 g for 3 h, aliquoted, and stored at −80°C.

The lentiviral vector pCDH-PGK-hPDGF-BB-mRFP for human PDGF-BB secretion was produced in human embryonic kidney 293 T cells (ATCC, Manassas) by co-transfecting with the human PDGF-BB over-expressing transfer plasmid, packaged plasmid (psPAX2), and enveloped plasmid (pMD2G VSVG) using a calcium phosphate transfection kit (Takara Bio USA, Inc., Mountain View, CA, USA) with 25 mmol/L chloroquine. The virus was mixed in a serum-free medium with 6 μg/mL of polybrene (Sigma Aldrich, St. Louis, MO, USA) and incubated at the MOI of 80 for 6 h to infect into previously established rabbit IL4-MSCs. The infected cells were confirmed to be GFP- or RFP-positive cells by fluorescence microscope on day 4 post-infection. IL4-PDGF-BB-MSCs from passages 8–10 were used after testing the IL4 and PDGF-BB secreting levels with ELISA. Thus, four kinds of MSCs were established in this study (Figure 1).

Primary neurons were transfected with pmaxGFP^TM (Lonza, Basel, Switzerland) at DIV 8 using a calcium phosphate transfection kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. On the appropriate DIV, primary neurons were fixed with 4% PFA and green fluorescence signals were visualized by confocal microscopy (Leica). The in vitro dendritic spine analysis was the same as the in vivo dendritic spine analysis. All experiments were performed in triplicate and at least 30 neurons were analyzed for each experimental group.

HEK293 and HeLa cells (ATCC, catalog nos. CRL-1573 and CL-0101, respectively) were cultured regularly in DMEM supplemented with 10% FBS (and 5% penicillin and streptomycin at 37 °C with 5% CO₂ (refs. ^{34 (link),35 (link)}). Transfection was performed by electroporation using the Bio-Rad Gene Pulser Xcell system. Transfected cells were seeded on round coverslips and cultured in OPTI-MEM medium containing 7% FBS. All experiments were carried out 24 h after transfection.
Hippocampal tissue from E18 Wistar rats was first digested with 0.1% trypsin at 37 °C for 15 min, washed with mixed complete medium (l-glutamine DMEM-F12 plus 10%FBS) and then pipetted with a Pasteur glass tube (15 mm) to get a cell suspension. Neurons were then plated at 50,000 cells per 18 mm coverslips coated with 0.1% poly-d-lysine and cultured in a 37 °C, 5% CO₂ incubator. Half of the medium was replaced with neurobasal medium (containing 2% B27 supplement, 0.5 mM GlutaMAX-100X) without serum 24 h later, and once per week thereafter. Arac (10 µM) was added 7 days after plating to inhibit gliacyte. Neurons were transfected with GCaMP6 or NEMO-encoding plasmids at 7–9 days after plating using a calcium phosphate transfection kit (Takara Bio).