HeLa transfection is usually pretty standard in terms of protocols - just make sure your nucleic acid concentration is conforming to that of the protocol you're using and you should be fine. Here's a protocol from a HeLa transfection kit (https://altogen.com/product/hela-transfection-reagent-cervix-adenocarcinoma-cells/) that I've pasted here in case you need it:

1. Plate 7,500 - 12,000 HeLa cells 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 serumfree medium, 5.5 µl of transfection reagent, and

• 750 ng DNA (or mRNA), or

• 30 nM - 50 nM of siRNA (or microRNA)

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 HeLa cells (from step 2)

7. Incubate cells at 37ºC in a humidified CO2 incubator 8. Assay for phenotype or target gene expression 48 - 72 hours after transfection

Hi, I am not quite sure why you are worried about the ratio of Opti-media to your lipofectamine. I thought you could just use as little as possible to increase the contact of DNA-Lipid complex with your cells while the cells will not dry out. But here is how I use lipofectamine 2000.

Note: I always prepare the quantity more than the standard protocol because layers in my incubator are not very horizontal and flat. Some of my cells will dried if I used the volume suggested by the manual.

The manual is here: https://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_2000_Reag_protocol.pdf)

1. Mix 2 ug DNA in 200 uL Opti-MEM.

2. Mix 6 uL lipofectamine 2000 in 200 uL Opti-MEM. (I usually start the DNA:Lipofectamine ration from 1:3 to 1:6. You can try to optimize the ratio for your cells.)

3. Mix 1 and 2 by adding the DNA to lipofectamins "DROP-WISE". (DO NOT MIX WITH OPPOSITE WAY.)

4. Incubate 5-10 min (make sure no more than 15 min).

5. Aspire culture medium from 6-well plate, wash with PBS twice and Opti-MEM once. Add 200 uL Opti-MEM (or complete medium) to each well.

6. Add the mixture dropwise to the well, the final vol should be 600 uL/well.

7. Incubate 8 h to overnight. (Lipofectamine is somewhat toxic to some cells, so make sure you find the optimized incubation time. Most of the time, I only incubate 8 h.)

8. Add 1 mL complete medium to each well. Just to make sure your cell will not dry out after transfection.

I usually change to fresh complete medium 18-24h post-transfection and select the cells by antibiotic containing medium after 48-72 h transfection.

p.s. It is HeLa cells, so if you do not have Opti-MEM, you can just use serum-free medium. Be sure you add 600 uL 20% serum-containing medium to each well at step 8 to make the cells cultured in 10% serum culture medium.

Good luck with your transfection : )

thanks allot for your suggestion . I will let you know for my success, I will be doing today my transfection.

Dear Jitendra,

Maybe I am not to late to present you an alternative to HeLa cells transfection ; we have actually launched a transfection reagent dedicated to this specific application named HeLaFect.

Its high compaction level of nucleic acid associated with low toxicity (biodegradable) make this reagent really efficient and easy to use (no need to change medium for example). More information are avaliable in the link below.

I would be very glad to send you a sample if you would like to try it; you can contact me via ResearchGate or directly at [email protected]

good luck with your experiment,

Cedric

http://www.ozbiosciences.com/transfection-cell-specific/77-hela-cells-transfection-helafect-reagent.html

How was your trial w Lipofectamine on HeLa cells? If you need further help I can send you a protocol via email. Please do let me know at sudas_1@yahoo

HeLa transfection is usually pretty standard in terms of protocols - just make sure your nucleic acid concentration is conforming to that of the protocol you're using and you should be fine. Here's a protocol from a HeLa transfection kit (https://altogen.com/product/hela-transfection-reagent-cervix-adenocarcinoma-cells/) that I've pasted here in case you need it:

1. Plate 7,500 - 12,000 HeLa cells 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 serumfree medium, 5.5 µl of transfection reagent, and

• 750 ng DNA (or mRNA), or

• 30 nM - 50 nM of siRNA (or microRNA)

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 HeLa cells (from step 2)

7. Incubate cells at 37ºC in a humidified CO2 incubator 8. Assay for phenotype or target gene expression 48 - 72 hours after transfection