1. Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Altogen Biosystems offers the A549 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. A549 Transfection Reagent is an advanced formulation of a lipid based reagent, and it has been developed to provide high transfection efficiency with A549 and other lung carcinoma cell lines. This cell line is a good host for studying lung cancer and human infections related to the lungs. When cultured in vitro, A549 cells are a monolayer and adhere to the surface of the culture flask. Other applications include drug discovery, gene expression studies, molecular and cell biology research applications. Purchase A549 Transfection Kit at www.Altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products www.Altogen.com Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

2. The A549 cell line comes from carcinomatous lung tissue of a 58 year old Caucasian male in 1972. These cells are squamous alveolar basal epithelial cells that grow as a monolayer and they are responsible for the diffusion of water and electrolytes in the lung alveoli. The cells also play a role in the synthesis of lecithin through the cytidine diphosphocholine pathway. The A549 cell line is hypotriploid and occurs in 24 percent of cells with the modal chromosome number being 66. A549 cells also frequently display chromosome counts of 64, 65, and 67. A549 cells are positive for keratin, as indicated through immunoperoxidase staining. This cell line is most often used for research related to respiratory infections, viral infections related to asthma, asbestos exposure and smoking-related emphysema. A549 Cell Line Description and Applications Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com www.Altogen.com Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

3. A549 Transfection Protocol 1. Plate 10,000 - 15,000 a549cells per well in 0.5 ml of complete growth medium 12–24 hours prior to transfection 2. Wash with 1xPBS and add 0.5 ml of fresh growth medium 3. Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µl of transfection reagent, and 750 ng DNA (or mRNA), or 30 nM - 50 nM of siRNA (or microRNA) *Referred to a final volume including growth medium 4. Incubate transfection complexes at RT for 15 - 30 minutes 5. Optional: Add 2 µl of Complex Condenser. This reagent reduces the size of transfection complex, therefore increasing transfection efficiency; however it may increase cell toxicity 6. Add prepared transfection complexes to 0.5 ml of complete growth medium with a549cells (from step 2) 7. Incubate cells at 37ºC in a humidified CO 2 incubator 8. Assay for phenotype or target gene expression 48 - 72 hours after transfection Optional: Transfection efficiency can be increased by addition of Transfection Enhancer reagent. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection If the viability of A549 cells being transfected is affected at 16 - 24 hours post-transfection, the level of cytotoxicity can be decreased by changing the growth medium and eliminating redundant exposure of cells to transfectant Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

4. General Lipoplex-mediated Transfection Mechanism of Action Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

5. A549 Transfection Kit Product Details Two component formulation enhances lipid mediated transfection efficiency Optimized easy-to-use transfection protocol provided for transfection of siRNA, DNA, mRNA, and microRNA Kit includes Transfection Enhancer reagent and recommended transfection protocol High transfection efficacy in the presence of serum Expand your RNAi application with a reagent optimized for delivery of both siRNA and plasmid Reproducible transfection results Works well for standard reverse transfection and high-throughput applications Developed and manufactured by Altogen Biosystems (www.Altogen.com) Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

6. Data Figure 1. SiRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected following the recommended protocol. At 48 hours post-transfection the a549cells were analyzed by qRT-PCR for gene expression levels. 18S rRNA levels were used to normalize the Lamin A/C data. Values are normalized to untreated sample. Data are means ± SD (n=3). Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

7. Data Figure 2. Protein expression of Lamin A/C in A549 cells. DNA plasmid expressing Lamin A/C or siRNA targeting Lamin A/C were transfected into A549 cells following Altogen Biosystems transfection protocol. At 72 hours post-transfection the cells were analyzed by Western Blot for protein expression levels (normalized by total protein, 10 µg of total protein loaded per each well). Untreated cells used as a negative control. Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

8. Pre-optimized transfection protocol for A549 cell line Compatible with DNA, small RNA (siRNA, shRNA, miRNA), mRNA, and small protein complexing Free of serum and protein of animal origin Compatible with standard and reverse transfection methods (both protocols provided in the kit manual) Easy to use A549 transfection protocol ensures great performance with expedited experimental timeline Equally efficient for single or multiple transfections Can be used for transient transfection and development of stable a549cell lines Bio-degradable after endocytosis Used for preclinical research worldwide A549 Transfection Kit Benefits Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > A549 Transfection Reagent (Lung Carcinoma, CCL-185) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products