protocol

Cells Transfected Successfully

CellLines CellType Species Reagents
3T3-F442A  Adipocytes  Mouse  Lipofectamine® Reagent 
SW13  Adrenal carcinoma  Human  Lipofectin® Reagent 
Primary  Amniocytes  Human  Lipofectin® Reagent 
T24  Bladder carcinoma  Human  Lipofectin® Reagent 
U-2 OS  Bone sarcoma  Human  Lipofectamine® 2000 Reagent 
KG-1; KG1a; K562  leukemia  Human  Lipofectamine® 2000 Reagent 
SK-BR-3  Breast Cancer  Human  Lipofectamine® 2000 Reagent 
MCF7  Breast Cancer  Human  Lipofectamine® 2000 Reagent 
Hs 578T; T-47D; MDA-MB-231; ZR-75-1  Breast Cancer  Human  Lipofectamine® Reagent 
C127  Breast Cancer  Mouse  Lipofectin® Reagent 
NMU  Breast Cancer  Rat  Lipofectamine® Reagent 
HeLa  Cervical Cancer  Human  Lipofectamine® 2000, Lipofectamine® Reagent 
C-33 A  Cervical Cancer  Human  Lipofectamine® Reagent 
HT-29  Colon  Human cancer  Lipofectamine® 2000 Reagent 
HCT116  Colon  Human cancer  Lipofectamine® 2000 Reagent 
COLO 320 HSR; SW40; 3SW480; Caco-2  Colon  Human cancer  Lipofectamine® Reagent 
T84  Colon  Human cancer  Lipofectin® Reagent 
Connective Tissue  Mouse  Lipofectamine® Reagent; Lipofectin® Reagent 
Isikawa  Endometrial cancer  Human  Lipofectamine® Reagent 
Primary HUVEC  Endothelial  Human  Lipofectin® Reagent 
Primary HUAEC  Endothelial  Human  Lipofectin® Reagent 
Primary Endothelial  Endothelial  Rabbit  Lipofectin® Reagent 
P19; F9  Embryonic carcinoma  Mouse  Lipofectamine Plus® Reagent 
Primary Fibroblast  Fibroblasts  Human  Lipofectamine® 2000 Reagent 
Mv 1 Lu  Lung Fibroblasts  Mink  Lipofectin® Reagent 
NIH 3T3; TA1; Y-2; NIH 3T6; 10T1/2  Fibroblasts  Mouse  Lipofectamine® with Plus™ Reagent, Lipofectin® Reagent 
MDCK  Kidney  Dog  Lipofectamine® Reagent 
BHK-21  Kidney  Hamster  Lipofectamine® with Plus™ Reagent 
293, 293H, 293F  Kidney  Human  Lipofectamine® 2000 Reagent 
A498; UMRC 6  Kidney carcinoma  Human  Lipofectamine® Reagent 
COS-1; COS-7; Vero CV-1  Kidney  Monkey  Lipofectamine® 2000 Reagent 
LLC-PK  Kidney  Pig  Lipofectamine® Reagent 
FLK  Kidney  Sheep  Lipofectamine® Reagent 
Primary Liver  Liver  Dog  Lipofectin® Reagent 
Huh7  Liver cancer  Human  Lipofectamine® 2000 Reagent 
Hep 3B  Liver cancer  Human  Lipofectamine® 2000 Reagent 
Primary Liver  Liver  Mouse  Lipofectin® Reagent 
Primary Liver  Liver  Rat  Lipofectamine® 2000 Reagent 
FTO-2B  Liver cancer  Rat  Lipofectamine® with Plus™ Reagent 
MSN610.2; Morris 7777; H35; McA-RH7777  Liver cancer  Rat  Lipofectamine® Reagent; Lipofectin® Reagent 
R1610  Lung  Hamster  Lipofectamine® with Plus™ Reagent 
Dede  Lung  Hamster  Lipofectamine® Reagent 
MRC-5  Lung  Human  Lipofectamine® 2000 Reagent 
WI-38  Lung  Human  Lipofectamine® Reagent 
NCI-H23  Lung  Human cancer  Lipofectamine® Reagent 
A549  Lung  Human carcinoma  Lipofectamine® 2000 Reagent 
H187; NCI-H146  Lung  Human small cell carcinoma  Lipofectin® Reagent 
LLC  Lung  Mouse carcinoma  Lipofectamine® Reagent 
BL3.1  Lymphoid  Bovine leukemia  Lipofectamine® Reagent 
Jurkat  Lymphoid  Human lymphoma  Lipofectamine® 2000 Reagent 
U937  Lymphoid  Human lymphoma  Lipofectin® Reagent 
MOLT-4  Lymphoid  Human leukemia  DMRIE-C Reagent 
A20.J; M12  Lymphoid  Mouse B cell lymphoma  Lipofectamine® Reagent 
P388D1  Lymphoid  Mouse lymphoma  Lipofectamine® Reagent 
THP-1  Macrophages/  Human monocyte  Lipofectamine® Reagent 
Primary; J774.16; RAW 264.7  monocytes  Mouse macrophage  Lipofectamine® Reagent; Lipofectin® Reagent 
B16  Melanoma  Mouse  Lipofectamine® 2000 Reagent 
Trachea smooth muscle  Muscle  Cow  Lipofectamine® Reagent 
C2C12  Muscle  Cow  Lipofectamine® with Plus™ Reagent 
Primary; G8  Muscle  Mouse  Lipofectamine® Reagent 
Primary  Muscle  Rat  Lipofectin® Reagent 
L6E9  Muscle  Rat  Lipofectin® Reagent 
A10  Smooth muscle  Rat  Lipofectamine® Reagent 
U-87; A-172  Glioma  Human  Lipofectamine® 2000 Reagent 
SK-N-MC  Neuroblastoma  Human  Lipofectin® Reagent 
SK-N-SH  Neuroblastoma  Human  Lipofectamine® Reagent 
Neuro-2a  Neuroblastoma  Mouse  Lipofectamine® Reagent 
Primary fetal astrocyte  Astrocyte  Rat  Lipofectamine® Reagent 
C6  Glioma  Rat  Lipofectamine® Reagent 
PC 12  Pheochromocytoma  Rat  Lipofectamine® 2000 Reagent 
CHO-K1  Ovary  Hamster  Lipofectamine® 2000 Reagent 
CT60  Ovary  Hamster  Lipofectamine® with Plus™ Reagent 
AS52; CHO-DXB11  Ovary  Hamster  Lipofectamine® with Plus™ Reagent 
SKOV3  Ovary cancer  Human  Lipofectamine® Reagent 
Primary Ovary  Ovary  Mouse  Lipofectin® Reagent 
Capan-2  Pancreas cancer  Human  Lipofectamine® Reagent 
AR4-2J  Pancreas  Rat  Lipofectamine® Reagent 
Primary fetal islet cells  Pancreas islet  Rat  Lipofectin® Reagent 
AtT-20  Pituitary  Mouse  Lipofectamine® Reagent 
Primary Pituitary  Pituitary  Rat  Lipofectin® Reagent 
GH3  Pituitary tumor  Rat  Lipofectamine® Reagent 
LNCaP; PC-3  Prostate  Human  Lipofectin® Reagent 
Primary keratinocytes  Skin  Human  Lipofectamine® 2000 Reagent 
AKR  Spleen  Mouse  Lipofectin® Reagent 
Sperm  Sperm  Mouse  Lipofectin® Reagent 
Primary hematopoetic CD34+  Stem cells  Human  DMRIE-C Reagent 
D3  Stem cells  Mouse  Lipofectin® Reagent 
SK-UT-1  Uterus  Human  Lipofectamine® Reagent 
Blastoderm cells    Chicken  Lipofectin® Reagent 
Primary chondrocytes    Chicken  Lipofectamine® Reagent 
Primary fibroblasts    Chicken  Lipofectamine® Reagent, Lipofectin® Reagent 
Primary hepatocytes    Chicken  Lipofectin® Reagent 
BMS-M protoplasts    Corn  Lipofectin® Reagent 
Dictyostelium      Lipofectin® Reagent 
A6 kidney cells  Xenopus laevis  Frog  Lipofectamine® with Plus™ Reagent 
Eggs; kidney cells  Xenopus laevis  Frog  Lipofectin® Reagent 
D.Mel-2; Schneider’s L2  Drosophila  Insect  Cellfectin® Reagent 
Sf9; Sf21; High Five™  Spodoptera frugiperda  Insect  Cellfectin® Reagent 
Neurospora crassa  Neurospora crassa  Insect  Lipofectin® Reagent 
DGZ protoplasts    Rice  Lipofectin® Reagent 
Primary lymphoid    Shrimp  Lipofectin® Reagent 
Protoplasts    Tobacco  Lipofectin® Reagent 
Saccharomyces cerevisiae  Saccharomyces cerevisiae  Yeast  Lipofectin® Reagent 
Schizosaccharomyces pombe  Schizosaccharomyces pombe  Yeast  Lipofectin® Reagent 
TOP

Troubleshooting

Transfection Problem Possible Cause Suggested Solution
Low transfection efficiency.  Not using optimal cationic lipid reagent.  Select the cationic lipid reagent likely to result in the highest transfection. efficiency for your cell type. See the references listed in the “Transfection Collection” on the web at www.invitrogen.com find the best reagent for your cell type. 
Low transfection efficiency.  Not using optimal cationic lipid reagent concentration, DNA concentration, time or cell density.  Optimize these parameters under final transfection conditions. See the cell-specific transfection protocols on the web at www.invitrogen.com
Low transfection efficiency.  DNA-cationic lipid reagent complexes not formed.  Do not use serum during complex formation step. Opti-MEM® I Medium or D-MEM are good media for complex formation. If using serum containing medium for transfections, form the complex in the absence of serum. Note: Do not use Opti-MEM® I Medium with Lipofectamine® with Plus™ Reagent or for insect cells. Sf-900 II SFM gives optimal results for Sf9 or Sf21 cells. For D. mel-S2 cells, use Drosophila-SFM. 
Low transfection efficiency.  Inhibitors were present.  Do not use antibiotics, EDTA, citrate, phosphate, RPMI medium, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare complexes. 
Low transfection efficiency.  Cationic lipid was frozen.  Do not use cationic lipid reagents that have been frozen. Store at +4°C. 
Low transfection efficiency.  Improper cell density.  Cell density should be 70% to 90% confluent at time of transfection. (30%-70% confluent if using Optifect™ Transfection Reagent) 
Low transfection efficiency.  Problems with the transfection assay.  Include a positive control for the transfection assay. 
Low transfection efficiency.  The promoter-enhancer of the transfected DNA is not recognized by the cell type.  Make sure the transfected DNA is compatible with the target cell type. 
Low transfection efficiency.  For Lipofectin® Reagent, no preincubation of cationic lipid with medium.  Preincubate Lipofectin® Reagent in medium 30 min. prior to addition of diluted DNA. 
Low transfection efficiency.  Diluted Lipofectamine® 2000 Reagent was incubated too long before mixing with diluted DNA.  For dilutions prepared in Opti-MEM® I Medium, be sure that the diluted Lipofectamine® 2000 Reagent is combined with the diluted DNA within 30 min. If D-MEM is used as the diluent, mix with the diluted DNA within 5 min. 
Low transfection efficiency.  Cell density too low for Lipofectamine® 2000 Reagent transfection.  Use cells at 90% confluency for Lipofectamine® 2000 Reagent transfections. 
High cell death (toxicity)  Too much DNA.  Perform a dose-response curve to determine the optimal amount of DNA. Include a cationic lipid reagent in the dose-response transfections, as DNA alone has a minimal effect on cell growth. 
High cell death (toxicity)  Too much cationic lipid reagent.  Perform a dose-response curve to determine the optimal amount of cationic lipid reagent. Include DNA with the cationic lipid reagent in the dose-response experiment, as cationic lipid reagent alone has a minimal effect on cell growth. 
High cell death (toxicity)  Too few cells.  Perform a dose-response curve to determine the optimal number of cells per transfection. Balance cell number with efficiency for your application. 
High cell death (toxicity)  Cell viability decreased without serum.  Use Opti-MEM® I medium. Reduce or omit the number of washes in serum-free medium. Use 5 to 10% serum in the transfection medium. Be sure to form complexes in the absence of serum. 
High cell death (toxicity)  For stable transfection, selection antibiotic added too soon.  allow at least 48 h for cells to express resistance before adding selective antibiotic. 
High cell death (toxicity)  Antibacterial agents were used during transfection.  Do not use chloroquine, penicillin, or streptomycin during transfection because cationic lipid reagents make cells more permeable. 
Transfection not reproducible  Confluency at the time of transfection varied.  Keep transfection parameters such as confluency and phase of growth consistent. 
Transfection not reproducible  Cells changed in culture.  If possible, thaw fresh cells or obtain new cell line. 
Cationic lipid reagent solution is cloudy.    For Cellfectin® DMRIE-C, and Lipofectamine® 2000 Reagents this is normal. Be sure to mix reagent (invert 5 to 10 times) prior to taking an aliquot for transfection. 
Cationic lipid reagent solution is cloudy.    For Lipofectin® the reagent may have been frozen or stored below +4°C. Do not use. 
Cationic lipid reagent solution is cloudy.    For Lipofectamine® Reagent, after dilution in Opti-MEM® I this is sometimes seen. This can be used for transfection. 
Precipitation of DNA-cationic lipid reagent complex.  Excess EDTA present.  Dissolve the DNA in water or in TE with the EDTA concentration <0.3 mm in the dilutEd DNA. 
Precipitation of DNA-cationic lipid reagent complex.  Excess precipitation of cationic lipid reagent-DNA observed in complex.  Ensure concentrations of cationic lipid reagent and DNA do not exceed recommended amounts in complex formation. 
Precipitation of lipid reagent.  Reagent may have been frozen or stored below +4°C.  Do not use. 
TOP
LT076