Journal: eLife

Article Title: Distinct expression requirements and rescue strategies for BEST1 loss- and gain-of-function mutations

doi: 10.7554/eLife.67622

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , PolyJet In Vitro DNA Transfection Reagent , SignaGen Laboratories , SL100688 , For cell transfection.

Techniques: CRISPR, Knock-Out, Generated, Knock-In, Derivative Assay, Recombinant, Plasmid Preparation, Gene Expression, Cloning, Control, Sequencing, Molecular Cloning, In Vitro, Transfection, Software, Patch Clamp

Journal: ACS Nano

Article Title: Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells

doi: 10.1021/acsnano.3c09608

Figure Lengend Snippet: Calcium concentration and the types of medium determine the size and quantity of CaP nanoparticles. (A–C) Different CaCl 2 concentrations caused various sizes and numbers of CaP particles. 35 mM CaCl 2 shows a great number of CaP nanoparticles with relative smaller sizes. (D,E) The example images show the transfection efficiency of pLL3.7/ Syn-Gfp under indicated CaCl 2 concentration. GFP-positive neurons were in green. The quantitative data represent the numbers of GFP-positive cells under indicated CaCl 2 concentration. (F–H) The size and quantity of CaP particles were changed by different media. Neurobasal (NB) medium has the most optimized condition to generate CaP nanoparticles. (I,J) The example images display the transfection efficiency of pLL3.7/ Syn-Gfp under various media. GFP-positive neurons were in green. The quantitative data display the numbers of GFP-positive cells under indicated CaCl 2 concentration. (K–M) The size and number of CaP nanoparticles were changed under different buffer systems. HBSS showed the most optimized condition to remove CaP nanoparticles. Over 1000 particles were analyzed from six different areas of three independent experiments. Statistic: one-way ANOVA (B, C, E, G, H, J, L, and M). Values represent the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The commercial transfection kits includes Cytofect Neuron Transfection Kit (Cell Applications, TF886 KS), DreamFect Transfection Reagent (OZBiosciences, DF40500), GenJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, SL100488), GenMute Transfection Reagent (SignaGen Laboratories, SL100568), Lipofectamine 3000 Transfection Reagent (Thermo Fisher, L3000001), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778075), Nupherin Transfection Reagent (Zneo, BML-SE225-0075), PolyJet Transfection Reagent (SignaGen Laboratories, SL100688), TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR6003), TransIT-LT1 Transfection Reagent (Mirus Bio, MIR2304), and TurboFect Transfection Reagent (Thermo Fisher, R0531).

Techniques: Concentration Assay, Transfection

Journal: ACS Nano

Article Title: Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells

doi: 10.1021/acsnano.3c09608

Figure Lengend Snippet: Teleofection delivers nucleic acid into primary neurons at different days in vitro (DIV) with desired efficiency. (A,B) The examples of images show the transfection efficiency of primary neurons under the indicated incubation times and CaP nanoparticle numbers. The transfected neurons were with GFP expression in green and neuronal marker MAP2 in magenta. (C,D) The quantitative data from A and B show the transfection efficiency of neurons under different transfection conditions. The transfection efficiency can be manipulated by change incubation time and loading volume of the teleofection mixture. (E) The representative images show the transfected neuron (GFP in green), glia cell marker (GFAP in red), and neuronal marker (MAP2 in magenta) at indicated time points in rat primary neuron culture. (F,G) The quantitative data of E show the transfection efficiency of neuron and glia at different DIV primary culture. (H) The quantitative data indicate the transfection efficiency of spiny and aspiny neurons under time-course manner. Data were analyzed from six different areas of three independent experiments. Statistic: one-way ANOVA (B and D) and two-way ANOVA (F and H). Values represent the mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The commercial transfection kits includes Cytofect Neuron Transfection Kit (Cell Applications, TF886 KS), DreamFect Transfection Reagent (OZBiosciences, DF40500), GenJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, SL100488), GenMute Transfection Reagent (SignaGen Laboratories, SL100568), Lipofectamine 3000 Transfection Reagent (Thermo Fisher, L3000001), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778075), Nupherin Transfection Reagent (Zneo, BML-SE225-0075), PolyJet Transfection Reagent (SignaGen Laboratories, SL100688), TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR6003), TransIT-LT1 Transfection Reagent (Mirus Bio, MIR2304), and TurboFect Transfection Reagent (Thermo Fisher, R0531).

Techniques: In Vitro, Transfection, Incubation, Expressing, Marker

Journal: ACS Nano

Article Title: Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells

doi: 10.1021/acsnano.3c09608

Figure Lengend Snippet: Teleofection enables versatile serial or cotransfection in primary neurons for diverse cellular biology research aims. (A,B) The flowchart shows the experiment design for serial transfection of indicated plasmids. The examples of images show the expression pattern of transfected neurons with GFP (green) or mCherry (red), and neurons were outlined by MAP2 (magenta). Under serial transfection, different nucleic acids can be uptaken by different neurons sequentially. The representative images show the neurons transfected with GFP followed by mCherry transfection at DIV10 and 11, respectively. The arrows indicate GFP and mCherry double positive neurons. (C) The quantitative data display that most of the neurons express only a single transfected gene, and only around 10% of neurons can uptake both transfected genes. (D) The representative images indicate that, based on serial-transfection stratagem, the synaptic region composed by two different neurons in green (dendritic spine) and red (axonal terminal) can be illustrated clearly. The thickness of a slice interval of a Z -stack is 0.5 μm. Also see Videos S9–S11 . (E) The flowchart shows the experiment design for serial transfection of Cmtr1 siRNA followed by pLL3.7/ Syn-Gfp transfection at DIV7 and 10, respectively. The neurons were fixed for ICC at DIV20. (F) The representative images show the neuron-expressed GFP (green) and mCherry (red) as the control and Cmtr1 knockdown group, respectively. The CMTR1 signal was displayed as magenta. (G) The high-magnification images from F show the details of transfected neurons. (H–J) The quantitative data from F and G represent CMTR1 intensity, dendritic number, and dendritic length in control and Cmtr1 siRNA knockdown groups. (K) The cartoon displays the definition and the pattern diagram of the dendritic branch types for image analysis. The quantitative data display the percentage of dendritic branch types in control and Cmtr1 siRNA-transfected neurons. Data were analyzed from at least six different areas of three independent experiments. Statistic: Student’s unpaired t test (H–K). Values represent the mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The commercial transfection kits includes Cytofect Neuron Transfection Kit (Cell Applications, TF886 KS), DreamFect Transfection Reagent (OZBiosciences, DF40500), GenJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, SL100488), GenMute Transfection Reagent (SignaGen Laboratories, SL100568), Lipofectamine 3000 Transfection Reagent (Thermo Fisher, L3000001), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778075), Nupherin Transfection Reagent (Zneo, BML-SE225-0075), PolyJet Transfection Reagent (SignaGen Laboratories, SL100688), TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR6003), TransIT-LT1 Transfection Reagent (Mirus Bio, MIR2304), and TurboFect Transfection Reagent (Thermo Fisher, R0531).

Techniques: Cotransfection, Transfection, Expressing

Journal: ACS Nano

Article Title: Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells

doi: 10.1021/acsnano.3c09608

Figure Lengend Snippet: Teleofection is applicable for molecular and biochemical analysis. (A) The flowchart shows the experiment design for cotransfection of pLL3.7/ Syn-Gfp and -mCherry at DIV7. Transfected neurons were harvested at DIV10 for Western blotting to detect the expression of GFP and mCherry. (B) The representative images show the protein expression level of delivery genes ( Gfp and mCherry ) under dose- and time-dependent manners in primary neurons. Two independent samples were displayed for each condition. Tubulin, actin, and GAPDH were detected as internal controls. (C) The flowchart shows the experiment design for Gfp mRNA transfection at DIV10, followed by sample collection at indicated times. (D) The representative image shows the expression level of GFP under time-dependent manners in primary neurons. (E) The flowchart indicates the experiment design for a dual-reporter assay in primary neurons. Firefly and Renilla Luciferase reporter plasmids were cotransfected into neurons at DIV7, and the reporter assay was performed at DIV10. (F) The expression of endogenous CPEB2 was detected by Western blotting. (G,H) The table represents the Luciferase activity of Firefly and Renilla in transfected neurons. The quantitative data were displayed as a dot plot graph in H. Data were analyzed from three independent experiments. Statistic: Student’s unpaired t test (H). Data were analyzed from three independent experiments. Values represent the mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The commercial transfection kits includes Cytofect Neuron Transfection Kit (Cell Applications, TF886 KS), DreamFect Transfection Reagent (OZBiosciences, DF40500), GenJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, SL100488), GenMute Transfection Reagent (SignaGen Laboratories, SL100568), Lipofectamine 3000 Transfection Reagent (Thermo Fisher, L3000001), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778075), Nupherin Transfection Reagent (Zneo, BML-SE225-0075), PolyJet Transfection Reagent (SignaGen Laboratories, SL100688), TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR6003), TransIT-LT1 Transfection Reagent (Mirus Bio, MIR2304), and TurboFect Transfection Reagent (Thermo Fisher, R0531).

Techniques: Cotransfection, Transfection, Western Blot, Expressing, Reporter Assay, Luciferase, Activity Assay

Journal: ACS Nano

Article Title: Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells

doi: 10.1021/acsnano.3c09608

Figure Lengend Snippet: Teleofection is a stratagem for protein delivery in cell lines and primary neuron culture. (A) The flowchart shows the experiment design to generate different CaP nanoparticles with distinct proteins for the cell delivery. Also see Supporting Information Figure S8 for the stratagem of protein cotransfection by teleofection. (B) The representative images showcase nanoparticles containing various fluorescent dye-conjugated antimouse antibodies (Ab), showing green for Ab-488, red for Ab-594, and magenta for Ab-647. (C) The representative images show the global amount of CTTNBP2-GFP (green) in COS7 cells after protein transfection by teleofection. The high magnification displays the changes of CTTNBP2-GFP in a COS7 cell under time-dependent manner. The bright field displays the details of cell morphology. (D–E) The dot plot graphs represent the quantitative data of transfection efficiency and the degradation rate of CTTNBP2-GFP in COS7 cells. (F) The example of images indicates the distribution of CTTNBP2-GFP (green) in COS7 cells under different cell cycle stages. Nuclei and stress fibers were outlined by DAPI (blue) and phalloidin (red), respectively. The colocalization of CTTNBP2-GFP and stress fiber was displayed as yellow. (G) The flowchart indicates the experiment design to deliver reporter gene and protein sequentially in primary neuron culture. (H) The representative images show the distribution of CTTNBP2-GFP (green) in primary neurons after 24 h of transfection. The low-power field displays the global view of the primary neuron labeled by mCherry (red). Nuclei were labeled by DAPI in blue. The high magnification reveals the detailed distribution of CTTNBP2-GFP in the dendritic shaft. The colocalization of CTTNBP2-GFP and mCherry was manifested as yellow. (I) The representative images show the distribution of NuMA-C-GFP (green) in primary neurons after 24 h of teleofection. The low-power field displays the global view of primary neurons labeled by mCherry (white); nuclei were displayed as red by DAPI. The high magnification shows the distribution of NuMA-C-GFP in nuclei; the colocalization of NuMA-C-GFP and the nucleus was manifested as yellow. Data were analyzed from six different areas of three independent experiments. Statistic: One-way ANOVA (D and E). Values represent the mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The commercial transfection kits includes Cytofect Neuron Transfection Kit (Cell Applications, TF886 KS), DreamFect Transfection Reagent (OZBiosciences, DF40500), GenJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, SL100488), GenMute Transfection Reagent (SignaGen Laboratories, SL100568), Lipofectamine 3000 Transfection Reagent (Thermo Fisher, L3000001), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778075), Nupherin Transfection Reagent (Zneo, BML-SE225-0075), PolyJet Transfection Reagent (SignaGen Laboratories, SL100688), TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR6003), TransIT-LT1 Transfection Reagent (Mirus Bio, MIR2304), and TurboFect Transfection Reagent (Thermo Fisher, R0531).

Techniques: Cotransfection, Transfection, Labeling

Journal: ACS Nano

Article Title: Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells

doi: 10.1021/acsnano.3c09608

Figure Lengend Snippet: Teleofection introduces an innovative method for transporting both nucleic acids and proteins into NSCs. (A) The flowchart shows the experiment design to deliver nucleic acid into NSCs by teleofection. (B,C) The dot plot graph represents the quantitative data of transfection efficiency of pLL3.7/ Syn-Gfp in NSCs. The representative images show the population of GFP-positive cells after teleofection. The high-power field exhibits the coexpression of NSC markers, Nestin and Notch1, in GFP-positive cells. (D) The example images show the expression level of GFP under dose-dependent manners by Western blotting after teleofection in NSCs. (E) The table represents the activity of Firefly and Rellina Luciferase in transfected NSCs. The quantitative data were displayed as a dot plot graph. Data were analyzed from four independent experiments. (F) The dot plot graph displays the transfection efficiency of CTTNBP2-GFP in NSCs by teleofection with a dose-dependent manner. (G) The representative images show the distribution of CTTNBP2-GFP (green) in NSCs after 24 h of teleofection. Nuclei and F-actin were labeled by DAPI and phalloidin in blue and red, respectively. The colocalization of CTTNBP2-GFP and F-actin was manifested as yellow. The orthogonal view of white squares from NSCs provides a comprehensive understanding of the relative distribution of CTTNBP2-GFP and F-actin from multiple angles. Statistic: One-way ANOVA (B and F). Student’s unpaired t test (E). Values represent the mean ± s.e.m., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The commercial transfection kits includes Cytofect Neuron Transfection Kit (Cell Applications, TF886 KS), DreamFect Transfection Reagent (OZBiosciences, DF40500), GenJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, SL100488), GenMute Transfection Reagent (SignaGen Laboratories, SL100568), Lipofectamine 3000 Transfection Reagent (Thermo Fisher, L3000001), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, 13778075), Nupherin Transfection Reagent (Zneo, BML-SE225-0075), PolyJet Transfection Reagent (SignaGen Laboratories, SL100688), TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR6003), TransIT-LT1 Transfection Reagent (Mirus Bio, MIR2304), and TurboFect Transfection Reagent (Thermo Fisher, R0531).

Techniques: Transfection, Expressing, Western Blot, Activity Assay, Luciferase, Labeling