hek293t cells aavpro 293t  (TaKaRa)

Journal: Nature Methods

Article Title: Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

doi: 10.1038/s41592-026-03023-y

Figure Lengend Snippet: ( a ) Concentration-dependent increase in refractive indices of candidate chemicals in water. ( b ) Calcium responses of GCaMP6f-expressing HEK293T cells to 50 μM ATP, measured using a plate reader. The refractive index was adjusted to 1.365 except for 5% glycerol (refractive index 1.348). Osmolarity was not adjusted to isotonicity (hypertonic, 300-700 mOsm/kg see also c ) in this experiment. After incubation with the clearing media for 4 hours, GCaMP6f responses were measured. n = 8 wells each. *** p < 0.001; ** p < 0.01; * p < 0.05; n.s., not significant ( p ≥ 0.05) (two-sided Dunnett’s multiple comparison test). ( c ) Calcium responses of GCaMP6f-expressing HEK293T cells to 50 μM ATP in the media with variable osmolality (300-700 mOsm /kg). The osmolality was adjusted with 10x PBS. The osmolalities of high MW media are also indicated. n = 8 wells each. ( d ) The osmolality of different BSA products in aqueous solution (refractive index 1.365, in ddH 2 O). n = 3 each. There are several products of BSA with different grades of purity are available from different companies. The osmolality was slightly different among these products, suggesting that salts remain in some of the products. ( e ) Residual salts contained in BSA products #1 and #2. The amounts of Na + , K + , Ca 2+ and Mg 2+ in BSA powder were measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The Cl − amount was obtained from the product’s certificate of analysis, which was measured by ion chromatography. Ionic concentrations of BSA solution at a refractive index of 1.365 (17% w/v) are shown. In the preparation of SeeDB-Live, residual salts contained in the BSA powder were taken into account. ( f ) Transmittance of live HeLa cell suspension (4 × 10 6 cells/mL, transmittance at 600 nm) in isotonic BSA#1/PBS at refractive index 1.365-1.375. n = 3 each. ( g ) Growth ratio of HeLa/Fucci2 cells in refractive index-optimized (refractive index 1.363, 320 mOsm/kg) medium of different BSA products. Cell numbers were counted based on the cell nuclei in the fluorescence images, and the number was normalized by the control data at each stage (n = 5 wells). Purification methods are not disclosed for most BSA products. ( h ) Growth curve of HeLa cells cultured in a 35 mm dish. Cell numbers in suspension was measured with a hemocytometer after trypsinization. The medium was isotonic, and their refractive indices were adjusted to 1.363. n = 3 dishes each. n.s.; not significant ( p ≥ 0.05) (Wilcoxon rank sum test corrected with Holm-Bonferroni correction). ( i ) Growth curve of HeLa/Fucci2 cells cultured in the inverted culture in 384-well plates. Cell number was counted based on fluorescence images of the nuclei. n = 5 wells. *** p < 0.001 (two-sided Dunnett’s multiple comparison test). ( j ) Standard curve for the viscosity measurement using sucrose solution. Flow speed was determined using 50 mL syringes. Plot was fitted with a single-exponential curve. n = 5 each. The viscosity of sucrose solution was obtained from the previous literature . Created in BioRender. Imai, T. (2026) https://BioRender.com/gyynf4j . ( k ) Viscosity of candidate solutions (in ddH 2 O). The viscosity was calculated based on the flow rate in ( j ). n = 5 each. ( l ) Specific gravity of the candidate solutions in PBS. The refractive indices of iodixanol and BSA#1 were 1.366. Specific gravity of cells are cited from previous studies . ( m, n ) Some of the BSA products have autofluorescence signals of unknown origin, especially for UV to blue range; therefore, we carefully selected low autofluorescence and low toxicity products from multiple suppliers. Absorbance ( m ) and fluorescence ( n ) of fetal bovine albumin (FBS), iodixanol, and BSA from different manufacturers are shown. Except for FBS, they are adjusted to 15% (w/v) in ddH 2 O. FBS was non-diluted. Data with error bars indicate mean ± SD. See Supplementary Table for detailed statistical data. PG: propylene glycol, BSA#1: BSA crystal (bioWORLD), BSA#2: BSA Low Salt (Fujifilm), BSA#3: BSA crystal (Fujifilm), BSA#4: BSA pH 5.2 (Fujifilm), BSA#5: BSA pH 7.0 (Fujifilm), BSA#6: BSA Globulin Free (Fujifilm), BSA#7: BSA Protease Free (Fujifilm).

Article Snippet: HEK293T cells (AAVpro 293T, 632273, Takara) were cultured in DMEM (high glucose) supplemented with 1% penicillin–streptomycin and 10% FBS at 37 °C and 5% CO 2 .

Techniques: Concentration Assay, Expressing, Refractive Index, Incubation, Comparison, Clinical Proteomics, Mass Spectrometry, Ion Chromatography, Suspension, Fluorescence, Control, Purification, Cell Culture, Viscosity

Journal: Nature Methods

Article Title: Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

doi: 10.1038/s41592-026-03023-y

Figure Lengend Snippet: a , Strategies for optical clearing of live cells. b , Transmittance (at 600 nm) of HeLa cell suspension (4 × 10 6 cells per ml) in isotonic saline solution with glycerol or iodixanol at different refractive indices. Fixed cells were treated with PFA and saponin. c , Calcium imaging of GCaMP6f-expressing HEK293T cells stimulated with 50 μM ATP. The refractive index of the medium was adjusted to 1.365 (except for 5% glycerol). Osmolarity was not adjusted to isotonicity. Data are the median ± interquartile range (IQR). *** P < 0.001; ** P < 0.01; NS, not significant ( P ≥ 0.05; two-sided Dunnett’s multiple-comparison test). d , The osmolality of candidate chemicals in aqueous solution (refractive index 1.365, in double-distilled water (ddH 2 O; n = 3 each). Sucrose was used as a control. Spherical polymers refer to polymers with highly branched and/or higher-order structure. BSA#1 and BSA#2 represent two examples of different BSA products. The osmolality of low-salt BSA (2) was 2.7 mOsm kg − 1 , consistent with its molar concentration (2.3 mM). e , The optimal refractive index of the extracellular medium was determined in PBS adjusted at different osmolalities. Transmittance of live HeLa cell suspensions (4 × 10 6 cells per ml) was measured. Left: optimal refractive index (1.369) of iodixanol-containing PBS. Right: optimal refractive index (1.363–1.369) of BSA-containing PBS ( n = 3 each). f , Phase contrast images of live HeLa cells in normal and BSA-containing medium (refractive index, 1.363). g , h , Growth of HeLa/Fucci2 cells. Cell numbers were measured by fluorescence imaging of cell nuclei ( n = 5 wells). g , Proliferation curve of HeLa/Fucci2 cells in iodixanol, Ficoll70 and BSA#1-containing medium (refractive index, 1.363; 320 mOsm kg −1 ). * P < 0.05; NS ( P ≥ 0.05; two-sided Dunnett’s multiple-comparison test). P values are <0.001 unless otherwise mentioned. h , Growth ratio in refractive index-optimized (refractive index, 1.363; 320 mOsm kg −1 ) medium compared to the control medium. * P < 0.05; NS ( P ≥ 0.05; two-sided Dunnett’s multiple-comparison test). P values are <0.001 unless otherwise mentioned. i – k , HeLa/Fucci2 cell spheroids cleared with SeeDB-Live. i , Phase contrast images of HeLa/Fucci2 cell spheroids under normal (left) and SeeDB-Live culture medium (refractive index 1.366, 320 mOsm kg −1 ; right). j , Growth curve of HeLa/Fucci2 cell spheroids with and without treatment with SeeDB-Live for 4 h per day. NS ( P ≥ 0.05; two-sided Wilcoxon rank-sum test combined with Holm–Bonferroni correction). k , Three-dimensional (3D) confocal images of a HeLa/Fucci2 cell spheroid. l , m , Intestinal organoids in Matrigel treated with SeeDB-Live (refractive index, 1.363) for 4 h per day. l , Phase contrast image. m , Growth of the intestinal organoids. The sizes of the organoids (areas in the phase contrast images) were determined using Cellpose. NS ( P ≥ 0.05; two-sided Wilcoxon rank-sum test combined with Holm–Bonferroni correction). n , 3D confocal images of GCaMP6s-expressing EECs in intestinal organoids from ePet-Cre; Ai162 mice before and after SeeDB-Live treatment. o , p , Calcium imaging of cortical organoids (confocal). Basal fluorescence (temporal median; left) and Δ F/F 0 images (right) of a cortical organoid labeled with a calcium indicator, Calblyte-650AM ( o ). Spontaneous calcium transients of neurons ( p ). q , Principles of optical clearing of live cells with SeeDB-Live. Maximum transparency was achieved by matching the refractive index of the extracellular medium to that of the cytosol (1.363–1.366). Data with error bars represent the mean ± s.d. Images are representatives of ≥3 trials. See Supplementary Table for detailed statistical data. MW, molecular weight; PG, propylene glycol; PEG, polyethylene glycol; HBCD, hyperbranched cyclic dextrin; PVP, polyvinyl pyrrolidone.

Article Snippet: HEK293T cells (AAVpro 293T, 632273, Takara) were cultured in DMEM (high glucose) supplemented with 1% penicillin–streptomycin and 10% FBS at 37 °C and 5% CO 2 .

Techniques: Suspension, Saline, Imaging, Expressing, Refractive Index, Comparison, Control, Concentration Assay, Fluorescence, Labeling, Molecular Weight

Journal: Nature Methods

Article Title: Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

doi: 10.1038/s41592-026-03023-y

Figure Lengend Snippet: ( a-g ) Long-term culture in SeeDB-Live. ( a ) Primary culture of cardiomyocytes. Phase contrast image of cardiomyocytes at DIV5 (top) and the timeline of the culture (bottom). AAV.PHP.S-CAG-jGCaMP8f-WPRE (2 × 10 10 gc/mL) was introduced at DIV1 and the medium was replaced at DIV2. Cells were cultured in SeeDB-Live medium (refractive index 1.366, 320 mOsm/kg) from DIV2 to DIV5. ( b ) Primary culture of cardiomyocytes before and after incubation in SeeDB-Live medium. Confocal image (top) and spontaneous calcium signals (bottom) at DIV2. ( c ) jGCaMP8f signals of cultured cardiomyocytes at DIV3 and DIV5. ( d ) Primary culture of hippocampal neurons. Phase contrast image (left) and the timeline of the culture (right). AAV-DJ-hSyn-jGCaMP8m-WPRE (7 × 10 10 gc/mL) was introduced at DIV2. Neurons were cultured in SeeDB-Live medium (refractive index 1.363, 230 mOsm/kg) from DIV7 to 10. ( e ) jGCaMP8m signals of cultured hippocampal neurons before and after incubation in SeeDB-Live medium. ( f, g ) Amplitude and frequency of spontaneous calcium signals for the control (black) and SeeDB-Live (red). n = 3 dishes each. n.s., non-significant (two-sided Wilcoxon rank sum test). ( h-k ) Comparison with other methods. ( h ) Transmittance of live HeLa cell suspension (4 × 10 6 cells/mL) at different wavelengths was compared among PBS, SeeDB-Live/PBS, and 0.6 M tartrazine/PBS . Due to high absorption of tartrazine, we could not compare transmission below 550 nm. ( i ) Phase contrast and fluorescence images of GFP-expressing HEK293T cells. The cells were immersed in Control (PBS), SeeDB-Live/PBS (refractive index 1.363, 296 mOsm/kg), and then in 0.6 M tartrazine/PBS (refractive index 1.43, 1407 mOsm/kg). Scale bar, 50 μm. ( j ) Phase contrast image of HeLa cells before and after immersion in 0.1 M tartrazine solution (RI 1.351), adjusted to isotonic condition (310 mOsm/kg). Scale bar, 50 μm. ( k ) Refractive indices, osmolalities, and transmittances (at 600 nm) were compared among clearing agents reported for live tissue 18,28 . Red indicates ideal ranges for non-invasive optical clearing of live cells. Data with error bars indicate mean ± SD. ( a ) - ( g ) show data from representative samples out of 3 trials. ( i ) and ( j ) show single-trial data. See Supplementary Table for detailed statistical data.

Article Snippet: HEK293T cells (AAVpro 293T, 632273, Takara) were cultured in DMEM (high glucose) supplemented with 1% penicillin–streptomycin and 10% FBS at 37 °C and 5% CO 2 .

Techniques: Cell Culture, Refractive Index, Incubation, Control, Comparison, Suspension, Transmission Assay, Fluorescence, Expressing