Application of poly-D-lysine in assisting suspension cells transfection and colony formation experiments
10.3760/cma.j.cn115356-20240131-00018
- VernacularTitle:多聚D-赖氨酸在辅助悬浮细胞转染及集落形成实验中的应用
- Author:
Jia LI
1
;
Jianpeng ZHOU
;
Ou BAI
Author Information
1. 吉林大学第一医院血液科，长春 130000
- Keywords:
Polylysine;
Suspension cells;
Transfection;
Clone formation
- From:
Journal of Leukemia & Lymphoma
2024;33(9):528-533
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the application of poly-D-lysine (PDL) in assisting suspension cells transfection and colony formation experiments.Methods:The human-derived diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-4 was selected. The cytotoxicity of different concentrations (0.01, 0.1, 1.0, 10.0 mg/ml) of PDL solution on SU-DHL-4 cells was assessed by using the CCK-8 assay, and cell viability was calculated. SU-DHL-4 cells with normal morphology in logarithmic growth phase were seeded into 96-well plates coated and uncoated with PDL. Cells were then infected with viral suspensions at a multiplicity of infection (MOI) of 50 or 100, and cell morphology was observed. A stable SU-DHL-4 cell model transfected with the luciferase gene was constructed by using lentiviral infection method. The infected SU-DHL-4 cells were divided into the control group (infected with control virus fluid) and the experimental group (infected with virus fluid carrying luciferase gene). The transfected cells were continuously cultured in RPMI 1640 complete medium containing puromycin. A dual-luciferase gene reporter assay kit was used to detect fluorescence intensity. Colony formation experiments with suspension cells were conducted by using PDL-coated dishes. The cells were cultured in RPMI 1640 complete medium containing 0.1% dimethyl sulfoxide (DMSO) (the control group), 0.1 μmol/L etoposide (group A), and 0.2 μmol/L etoposide (group B), and colony formation was observed and colony formation rate was calculated.Results:After 48 h of seeding SU-DHL-4 cells into 96-well plates coated with 0.01, 0.1, 1.0, and 10.0 mg/ml PDL, cell viability was (98.1±1.6)%, (97.1±0.7)%, (91.7±1.5)%, and (83.3±2.0)%, respectively, compared to (100.0±2.7)% in the control group (not adding PDL solution). There were no statistically significant differences between 0.01 and 0.1 mg/ml PDL-coated groups and the control group (both P > 0.05); there were statistically significant differences between 1.0 and 10.0 mg/ml PDL-coated groups and the control group (both P < 0.001). There were statistically significant differences between 1.0 mg/ml and 10.0 mg/ml PDL-coated groups ( t = 5.80, P = 0.004). Therefore, 0.1 mg/ml PDL-coated dishes, which showed no obvious toxicity to SU-DHL-4 cell growth, were used in subsequent experiments. SU-DHL-4 cells seeded in uncoated dishes grew in suspension, while SU-DHL-4 cells seeded in 0.1 mg/ml PDL-coated dishes adhered to the dish bottom and exhibited a monolayer growth pattern. SU-DHL-4 cells seeded in uncoated 96-well plates and infected with viral suspensions at MOI 50 or 100 failed to transfect. However, when SU-DHL-4 cells were seeded in PDL-coated 96-well plates, the originally suspended cells adhered to the dish bottom and exhibited a monolayer growth pattern, and gentle shaking did not dislodge the cells, which maintained intact and smooth cell membrane structures. After puromycin selection, the fluorescence intensity of SU-DHL-4 cells in the experimental group was 106 times that of cells in the control group (26 903±248 vs. 252±11, t = 186.10, P < 0.001), indicating successful transfection. Compared to the control group, colony formation was reduced and smaller in group A, and nearly no colony formation was observed in group B. The colony formation rates in the control group, group A, and group B were (100±6)%, (48±5)%, and 0, respectively, and the differences were statistically significant between groups A and the control group, between group B and the control group ( t = 11.13 for group A, t = 28.53 for group B, both P < 0.001). Conclusions:PDL-coated dishes at a concentration of 0.1 mg/ml can increase the transfection efficiency of suspension cells and assist in colony formation experiments with suspension cells.
