I try to transfect endothelial cells with firelfy luciferase plasmids under control of various promoters with Lipofectamine LTX, but transfection does not work at all, does any one have any idea how to transfect these endothelial cell lines ?
Hi,
we have experienced challenges in transfecting HUVECs and other primary human endothelial cells in the past. Using a transfection reagent specifically designed for these cells (we used PromoFectin-HUVEC) and removing the reagent after 4-6 hours solved the issue for us. Unfortunately I dont have any advice or experience regarding Human liver sinusoidal endothelial cells.
Best regards!
The Targefect-HUVEC reagent works very well for transfection of HUVECs and microvascular endothelial cells. Transfection efficiency in HIVECs is between 70-90% and there is an extensive list of citations . You can view product details on the following links
https://www.targetingsystems.net/pdf/Targefect_HUVEC.pdf
Article Transfection Protocol for Human Umbilical Vein Endothelial C...
The catalog no is HUVEC-01 I have also attached the product brochure
( product listed a the following link) https://www.targetingsystems.net/product-price-list.php#genedelivery. Hope this helps
Hi Vera,
I used promofectin HUVEC, it gave results which are above the control but the overall transfection is weak somehow. after how long did you analyse luciferase activity post transfection-3days or less?
Using Targefect-HUVEC you definitely still see very good activity at 3 days post transfection. Also transfection efficiency assessed by GFP is very high with for HUVECs and MVE. BTW what luciferase reporter are you using? Usually for primary cells such as HUVECs I prefer to use secreted reporters such as Gaussia luciferase or Cypridina luciferase becasue then you dont need to lyse cells and can take multiple time points
Hi Rampyari,
thank you for your advice, however, i have to use 2.5 ug of DNA in each well of a 6 well plate, your protocol only allows 1 ug for 6 well plate, is there any possible way that i can use 2.5 ug /well?
Hi Rampyari,
I use firefly luciferase, Could you advise me regarding the scaling up of the transfection that is to use 2.5 ug DNA /well in a 6 well plate?
Please scroll to Protocol 2 and look at TABLE 2 on page 3 and use condition 2 for complex formation. If you read the details on the fast transfection protocol on page 4 (pasted below) you will find that 250 ul transfection complex has about 2.5 ug DNA which is what you woudl like to use so this protocol will definitely work for you. BTW which firefly luciferase assay reagent are you using?
Condition 2 (complexes formed in tube 2) is for transfecting cells in the presence of serum according to our fast protocol.-250 µl of transfection complex is added to 1 ml of complete media (with serum) per well of a six-well dish. The dish is swirled to enable mixing of the transfection complex with the cell culture medium and the cells are incubated at 37°C overnight and assayed for gene expression 36-48 hours post transfection.
Add 0.5 ml of transfection complex to 2 ml of complete media with serum for one 60 mm dish. For transfecting cells in a 12-well dish add 0.125 ml transfection complex to 0.5 ml of complete media.
Swirl the dish to gently mix transfection complexes with the cell culture media. Incubate overnight. Replace media the next day. Assay at 24-48 hours after transfection.
it is as in-house made reagent
D-luciferin + ATP + Gly Gly + Magnesium sulphate
Hi Rampyari,
I wanted to order HUVEC01 from your company but they told our purchase department we have have to buy 2 pieces of this artikel, I just need one for now since i will just try to see if it works for me or not, can I just order one piece this time?
Yes you can. Please tell your ordering department to send an email saying the order form Ahmed Abdelrahman in the subject line and reference your contact info (email) on the order since we also email some important supporting material if needed
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