Transfection for HMECs utilizing Lipofectamine LTX + Plus reagent NB: • Using PLUS™ Reagent ( Cat. No. 11514-015 ) enhances transfection public presentation in HUVEC cells. • The add-on of antibiotics to media during transfection may ensue in cell decease
Transfection of HMECs
Use this process to transfect plasmid DNA into HMECs cells in a 12-well format
All sums and volumes are given on a per good footing.
1. Cell denseness should be 50~80 % feeder on the twenty-four hours of transfection ( use the normal growing medium without antibiotics ) . 2. For each well of cells to be transfected. thin 1 ?g of DNA into 200 ?l of Opti-MEM® Medium without serum. 3. Mix PLUS™ Reagent gently earlier usage. so add 1 ?l PLUS™ Reagent ( a 1:1 ratio to DNA ) straight to the diluted DNA. Mix gently and incubate for 5-15 proceedingss at room temperature. 4. For each well of cells. thin 4 ?l of Lipofectamine™ LTX into the above diluted DNA solution. blend gently and incubate for 25 proceedingss at room temperature to organize DNA-Lipofectamine™ LTX composites. 5. Remove growing medium from cells and replace with 1 milliliters of complete growing medium without antibiotics. Add 200 ?l of the DNA-Lipofectamine™ LTX composites straight to each well incorporating cells and blend gently by swaying the home base back and Forth. 6. Complexs do non hold to be removed undermentioned transfection. Incubate the cells at 37°C in a CO 2 brooder for 18-24 hours post-transfection before assaying for transgene look.