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HeLa S3 transfection with microparticle loaded pDNA - (Sep/01/2006 )

Hello,

this is the first time I am posting, so please bare with me.

I am trying to transfect my HeLa cells with poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with pGFP plasmid DNA but have problems.

Since I don't have much experience working with cells (I've recently transfered to cell biology from genetic engineering), I have couple of questions that I need answers.

1. The HeLa cells that I receieved were grown in DMEM (1X) (with L-Glutamine, 4500 mg/L D-Glucose, 110 mg/L Sodium Pyruvate) + 10% FBS (Don't know whether heat inactivated or not) + 5% Antibiotic/Antimycotic solution (100X). So I used the same medium and I had a less growth in the cells, as well as no transfection. What's going on?

2. Cells tend to accumulate in the middle of the 24-well plate. How can I get them to spread?

3. When I transfect with the particles, the particles tend to aggregate forming a white spot in the 24-well plates. How can I get ride of it?

4. I have heard that for transfection, the medium should not contain antibiotics? Is that true?

5. Do I have to heat inactivate my FBS before adding to the medium? What difference does it make?

6. When I transfect my cells, I do it while the cells are in the medium (DMEM + FBS + Anti), is that good or bad?

7. If I grow the cells in DMEM + 10% FBS + 5% Anti and when I split them into 24-well plate and then grow them in the well plate with DMEM + 10% FBS and without Antibiotics at all would that contaminate my cells? And is it good for the cells to have a sudden change in the medium like that?

8. I prepare particles in my working bench, in the normal lab, not under a hood. Is it better to prepare them under the hood? When I work in the bench I sterile it with EtOH 70%. Wouldn't it be enough?

I know it is a lot of questions, but please I really need to get this right. So any help would be appreciated. Thanks a lot.

-iceman009-

QUOTE (iceman009 @ Sep 1 2006, 02:30 AM)
Hello,

this is the first time I am posting, so please bare with me.

I am trying to transfect my HeLa cells with poly(lactic-co-glycolic acid) (PLGA) microparticles loaded with pGFP plasmid DNA but have problems.

Since I don't have much experience working with cells (I've recently transfered to cell biology from genetic engineering), I have couple of questions that I need answers.

1. The HeLa cells that I receieved were grown in DMEM (1X) (with L-Glutamine, 4500 mg/L D-Glucose, 110 mg/L Sodium Pyruvate) + 10% FBS (Don't know whether heat inactivated or not) + 5% Antibiotic/Antimycotic solution (100X). So I used the same medium and I had a less growth in the cells, as well as no transfection. What's going on?
No need to heat inactivate FBS.
Medium seems to be ok.
slow growth compare to what condition?
this formulation is known to be not very efficient. you may use other easy to transfect cell type, such as 293 to start with before your confidence runs out.

2. Cells tend to accumulate in the middle of the 24-well plate. How can I get them to spread?
I had similar problem before. Try to use large amount of medium, add it slowly toi the wells, return to incubator without too much shake.

3. When I transfect with the particles, the particles tend to aggregate forming a white spot in the 24-well plates. How can I get ride of it?
This type of particles are not very hydrophilic and tend to aggregate in medium.

4. I have heard that for transfection, the medium should not contain antibiotics? Is that true?
Not always. Some believe thats the case some dont.
5. Do I have to heat inactivate my FBS before adding to the medium? What difference does it make?
not much
6. When I transfect my cells, I do it while the cells are in the medium (DMEM + FBS + Anti), is that good or bad?
where else would you put your cells in? do you mean medium without serum? We do that with cationic lipid transfection
7. If I grow the cells in DMEM + 10% FBS + 5% Anti and when I split them into 24-well plate and then grow them in the well plate with DMEM + 10% FBS and without Antibiotics at all would that contaminate my cells? And is it good for the cells to have a sudden change in the medium like that?
It depends on your technique. You catch contamination by accident.

8. I prepare particles in my working bench, in the normal lab, not under a hood. Is it better to prepare them under the hood? When I work in the bench I sterile it with EtOH 70%. Wouldn't it be enough?
yes. if you are doing this without antibiotics.

I know it is a lot of questions, but please I really need to get this right. So any help would be appreciated. Thanks a lot.

Good luck!

-genehunter-1-

thanks genehunter for the reply. Yes, I meant the medium without serum biggrin.gif. So I suppose it doesn't make a difference whether I put with heat inactivated FBS or not, that's good to know.

When I spoke to some ppl in my lab, they told me to use DMEM with GlutaMax? what's so great about that?

This is my last question, is it o.k that I change the antibiotic concentration in my medium? What type of effects would that have on my cells other than the contamination possibility might increase?

Please let me know, because I want to have a proper medium and condition this time before I start doing the experiment. Thanks

-iceman009-

I meant we dont add serum when we do transfection with liposomes. We add complet medium with serum after transfection for 4 hrs. However, we use a system that is different from yours.

GluMax and Glutamine essentially do the same thing. GluMax has longer halflife.

I have not been doing changes with antibiotic concentration that much.

-genehunter-1-

if u want the cells to spread evenly in a 24 well plate with 500ul of media, tilt left to right - 90 degrees and then other side , for 10 times . Now do it front to back and forth -10 times. repeat left to right - 5 times and place plate into incubator carefully.

-scolix-