Fig 1.
Comparison of chemical transfections.
Fig 2.
Chemical transfection protocols.
Fig 3.
Fluorescence images of HEK 293T cells co-transfected with eGFP together with CaV2.2 subunits (α1B, α2δ1 and β1) cDNA using (A) calcium phosphate, (B) FuGENE or (C) Lipofectamine 3000 was used to monitor transfection efficiency. (D) The transfection efficiency of the calcium phosphate-, FuGENE- and Lipofectamine 3000-mediated transfection was 49.0 ± 3.1%, 58.7 ± 3.5% and 51.7 ± 4.9%, respectively. Data are represented as mean ± SEM (N = 3 independent experiments conducted in triplicate).
Fig 4.
The optimization of transient transfection.
(A) The comparison of 90 mM KCl-evoked fluorescence response from HEK293T cells transfected with CaV2.2, incubated in four different temperature conditions. The response over baseline from untransfected cells was 0.12 ± 0.0, which was significantly lower compared to transfected cells. The response over baseline from transfected cells incubated 72h at 37°C, 8h at 28°C plus 64h at 37°C, 16h at 28°C plus 56h at 37°C, and 24h at 28°C plus 48h at 37°C were 0.5 ± 0.0, 0.6 ± 0.0, 1.2 ± 0.0, and 1.0 ± 0.1. (B) The concentration response curve of KCl-evoked Ca2+ response from transfected and untransfected HEK 293T cells, EC50 = 39.7 ± 0.3 mM for transfected cells. (C) The concentration response curve of KCl-evoked Ca2+ response from transfected and untransfected COS-1 cells, EC50 = 68.7 ± 1.2 mM for transfected cells. (D) The concentration response curve of KCl-evoked Ca2+ response from transfected and untransfected CHO-K1 cells, EC50 = 42.5 ± 2.2 mM for transfected cells. Transfected COS-1 and CHO-K1 cells gave similar response to the corresponding untransfected cells due to low expression level. Data are represented as mean ± SEM (N = 3 independent experiments conducted in triplicate), *** and **** denotes P < 0.001 and P < 0.0001, respectively.
Fig 5.
Representative Ca2+ fluorescence responses for CaV2.2 (A, C, E) and NaV1.7 (B, D, F), transfected by calcium phosphate-, FuGENE-, and Lipofectamine 3000 measured in a FLIPRTETRA. The KCl-evoked Ca2+ fluorescence responses of CaV2.2 over baseline for calcium phosphate-, FuGENE-, and Lipofectamine 3000 were 1.3 ± 0.0 (A), 1.7 ± 0.1 (C), and 1.5 ± 0.1 (E), respectively. The addition of 500 nM, and 16 nM CVID blocked the fluorescence response by 99.4 ± 1.8%, and 51.2 ± 3.3%, respectively, for calcium phosphate (A), 100.8 ± 0.9% and 50.1 ± 1.2%, respectively, for FuGENE (C), and 99.1 ± 1.0% and 49.5 ± 2.1%, respectively, for Lipofectamine 3000 (E). The veratridine-evoked Ca2+ fluorescence responses of NaV1.7 over baseline for calcium phosphate-, FuGENE-, and Lipofectamine 3000-mediated transfetion were 0.9 ± 0.0 (B), 1.2 ± 0.1 (D), and 1.1 ± 0.1 (E), respectively. Addition of 1 μM, and 111 nM TTX blocked the fluorescence response by 98.9 ± 1.8% and 53.8 ± 1.6%, respectively, for calcium phosphate (B), 99.8 ± 0.4% and 54.2 ± 2.0%, respectively, for FuGENE (D), and 99.4 ± 1.1% and 51.2 ± 2.3%, respectively, for Lipofectamine 3000 (F). Data are represented as mean ± SEM (N = 3 independent experiments conducted in triplicate).
Fig 6.
(A) Representative concentration response curves of the KCl-evoked CaV2.2- responses in HEK 293T cells transfected by calcium phosphate (EC50 38.1 ± 2.3 mM), FuGENE (EC50 36.1 ± 0.3 mM) and Lipofectamine 3000 (EC50 39.7 ± 0.3 mM). (B) Representative concentration response curves of CVID inhibition of 90 mM KCl-evoked CaV2.2 responses in HEK 293T cells transfected by calcium phosphate (IC50 19.5 ± 0.6 nM), FuGENE (IC50, 15.2 ± 1.6 nM) and Lipofectamine 3000 (IC50 15.5 ± 1.1 nM). (C) Representative concentration response curves of veratridine-evoked NaV1.7 responses in HEK 293T cells transfected by calcium phosphate (EC50 28.6 ± 0.4 μM), FuGENE (EC50 28.9 ± 0.7 μM) and Lipofectamine 3000 (EC50 29.9 ± 1.0 μM). (D) Representative concentration response curves of TTX inhibition of 40 μM veratridine evoked activation of NaV1.7 in HEK 293T cells transfected by calcium phosphate (IC50 98.1 ± 3.4 nM), FuGENE (IC50 95.4 ± 7.0 nM) and Lipofectamine 3000 (IC50 101.7 ± 5.4 nM). Data are represented as mean ± SEM (N = 3 independent experiments conducted in triplicate).
Fig 7.
Calcium current measured with a QPatch.
Representative ICa during 120 ms depolarization to Vmax (0 mV) from a holding potential of –100 mV before and after perfusion of 20 nM and 250 nM CVID obtained from HEK 293T cells transfected by (A) calcium phosphate, (C) FuGENE or (E) Lipofectamine 3000. Representative INa during 20 ms depolarization to Vmax (0 mV) from a holding potential of –80 mV and pre-pulse of –120 mV before and after perfusion of 200 nM and 1 μM TTX obtained from HEK 293T cells transfected by (B) calcium phosphate, (D) FuGENE or (F) Lipofectamine 3000. The current amplitude of both FuGENE-, and Lipofectamine 3000-mediated transfection methods was significantly higher compared to calcium phosphate-mediated transfection for both CaV2.2 and NaV1.7 channels (G). Data are presented as mean ± SEM, from N = 3 independent experiments, each testing 2‒6 cells, *** and **** denotes P < 0.001 and P < 0.0001, respectively.
Fig 8.
Current-voltage relationship of CaV2.2 in the presence and absence of 20 nM CVID from HEK 293T cells transfected by (A) calcium phosphate, (C) FuGENE and (E) Lipofectamine 3000. CaV2.2-mediated currents were evoked by depolarization steps from a holding potential of –100 mV followed by –60 mV to +50 mV in 5-mV increment. Current-voltage relationship of NaV1.7 in the presence and absence of 200 nM TTX obtained from HEK 293T cells transfected by (B) calcium phosphate, (D) FuGENE and (F) Lipofectamine 3000. NaV1.7-mediated currents were evoked by step depolarization from a holding potential of –80 mV followed by –60 mV to +55 mV in 5-mV increment. Data are presented as mean ± SEM, with N = 4–9 cells per data point.
Table 1.
Success rates of different transfection methods in the QPatch 16X showing mean ± SEM from N = 3 independent experiments, each testing 8 cells (24 cells per total).