Miniaturization of gene transfection assays in 384- and 1536-well microplates

Abstract

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35μl and in 1536-well plates in a total assay volume of 8μl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.

Keywords: 1536-well; 384-well; Gene delivery; Gene transfection; Luciferase assay; Mammalian cell culture.

Figure 1

Luciferase Standard Curve in 384 and 1536-Well Plates. Panel A illustrates the linearity of response as a function of increasing amounts of luciferase and increasing luciferin (ONE-Glo) added to plated HepG2 cells in a 384-well plate. The results are the mean and standard deviation of n = 3. Panel B illustrates the linearity of response and dynamic range in a 1536-well plate (n = 4).

Figure 2

Transfection of HepG2 Cells in 384 and 1536-Well Format. Panel A illustrates the result of 384-well transfection of 5000 HepG2 cells in 25 μL media. At 48 hr following the addition of PEI gWiz-Luc (250 ng, N:P = 9:1) in 5 μL, 5-30 μL of ONE-Glo was added, and RLU were determined after 5 min (n = 3). Panel B illustrates transfection of 1200 HepG2 cells in 6 μL media in 1536-well plates, with 80 ng (2 μL) of PEI/gWiz-Luc (N:P = 9). At 48 hr after post-transfection, 1-3 μL of ONE-Glo was added prior to measuring RLU after 5 min (n = 4). * indicates p ≤ 0.05 relative to blank.

Figure 3

Influence of Cell Plating Number on Transfection in 384 and 1536-Well Format. Panel A illustrates the result of plating HepG2 cells (25 μL, 2500-20000 cells) in 384-well plates and transfecting with 5 μL (0-300 ng, PEI gWiz-Luc, N:P = 9). At 48 hr post-transfection, 10 μL of ONE-Glo was added and the RLU was measured after 5 min (n = 3). Panel B illustrates the result of transfection of 0-120 ng (2 μl) of PEI gWiz-Luc (N:P 9) into HepG2 cells (600-1500 cells in 6 μL per well) in 1536-well plates. At 48 hr, 3 μL was removed and 2 μL of ONE-Glo was added the bioluminescence data was measured at 25 min (n = 4). * indicates p ≤ 0.05 relative to 0 DNA dose.

Figure 4

Influence of Varying PEI to DNA Ratio and Bioluminescence Acquisition Time on Gene Transfer Efficiency. Panel A illustrates the result of transfecting 250 ng PEI gWiz-Luc with N:P ranging from 0-27 onto 5000 HepG2 cells per well in 384-well plates. Following the addition of 10 μL of ONE-Glo, bioluminescence was measured at a time varying from 5-60 min (n = 3). Panel B illustrates the result of transfecting 75 ng per well of PEI gWiz-Luc (2 μl) with N:P ranging from 0-26 onto 1200 HepG2 cells per well in 1536-well plates. At 48 hr following transfection, 3 μL was removed and 2 μL ONE-Glo was added followed by bioluminescence measurement at 5, 10, 25, 45 and 60 min (n = 4). An N:P of 0 indicates a transfection control with naked DNA which fails to mediate luciferase expression. * indicates p ≤ 0.05 relative to 0 N:P.

Figure 5

Whole Plate Transfection in 384 and 1536-Well Plates. The result of applying an optimized transfection protocol to HepG2 cells in 384 (Panel A) and 1536-well (Panel B) format are illustrated.

Figure 6

Transfection of HepG2 Cells with GFP in 384 and 1536-Well Plates. Panel A illustrates the result of transfection of 10,000 cells (25 μL) per well in 384-well plates with transfection with 250 ng (5 μL) of PEI gWiz-GFP (N:P 0-27). At 48 and 72 hr the fluorescence was measured at excitation 480 and emission 510 nm. Panel B illustrates the result of transfecting 1500 HepG2 cells (6 μl) in 1536-well plates with 60 ng (2 μL) of PEI gWiz-GFP (N:P 0-27) followed by fluorescence intensity analysis at 48 and 72 hr. An N:P of 0 indicates a transfection control with naked DNA which fails to mediate GFP expression.* indicates p ≤ 0.05 relative to 0 N:P.

Figure 7

Transfection of Primary Hepatocytes in 384-Well Plates. Panel A illustrates the transfection of 0-1000 primary hepatocytes in 45 μL in a 384-well plate with 400 ng (5 μL) of PEI gWiz-Luc (N:P 7). At 24 hr, 10 μL of ONE-Glo was added, followed by measurement of bioluminescence at 5 min. (n = 9). Panel B illustrates transfection of 1000 primary hepatocytes as described in panel A, with 400 ng (5 μl) of PEI gWiz at (N:P 0.5 to 13). Panel C illustrates the transfection of 250 primary hepatocytes per well with 400 ng of PEI gWiz-Luc (N:P 7) with bioluminescence measured every 12 hours for 96 hours (n = 9). * indicates p ≤ 0.05 relative to 0 DNA dose.

Figure 8

PEI and CaPO₄ Transfection of Primary Hepatocytes in 384-Well Plates. Panel A illustrates the transfection of 250 primary hepatocytes (45 μL) per well with 0-750 ng of PEI gWiz-Luc (N:P 7). At 24 hr 10 μL of ONE-Glo was added and bioluminescence was measured at 5 min (n = 9). Panel B illustrates the transfection of primary hepatocytes (45 μL) plated in 384-well plates at 250 cells per well with 250 ng of CaPO₄ calcium phosphate. At 24 hr 10 μL of ONE-Glo was added and bioluminescence was measured at 5 min (n = 9). * indicates p ≤ 0.05 relative to 0 DNA dose.