Keep doing what you're doing! My only concern would be contamination with repeated use of the aspirating pipet, but if you are careful not to touch the pipet to non-sterile surfaces, I see no problem in your procedure.

Dear David Belair,

I appreciate for your answer deeply!

If you don't mind, can i ask several additional questions?

What you expressed as 'non-sterile surfaces' are huvec cells, right?

And yes, I am always careful to prevent contact between suction tip(aspirating pipet tip) and huvec.

But If such contact occurred during my work without recognizing,

May I know what troubles happen?

Contamination? Any other risks? As for contamination, I rarely experienced contamination

but i want to make sure which problems including contamination happen when

huvec cells are touched by aspirating pipet tip.

Thank you

You can add a small tip to the suction pipette (to reduce speed) and use the suction carefully. 

I think what David meant by "non-sterile surfaces" are things like your hands/glove/labcoat, the surfaces of the biosafety cabinet, the exterior surfaces of bottles/tubes etc.

It doesn't matter if your pipet tip touches the huvec cells, other than possibly scratching up a few attached cells. What you have to be careful about is making sure that when you move or put down the suction tool, the tip doesn't touch anything else.

You also have to make sure that anything that touches your huvec cells or the medium they are in doesn't then touch your reagent stocks (like bottle of medium, trypsin etc). I think you already indicated yet, but it is not very clear because you mention both a pipet aid and a suction tool. Are you using a separate vacuum line to aspirate medium (what you call suction tool) or are you using your pipet aid to aspirate?

Dear Dmitriy Sheyn,

Thank you very much for your answer!

Good luck for your research!

Dear Shanshan Wu Howland,

I appreciate deeply for your answer!

I use pipet aid for draining medium or trypsin, not for suctioning. I use aspirating pipet and I anchor pipet tip at the end ot the aspirating pipet.

If you don't mind, can i ask several questions additionally?

In my procedure of huvec work described above, I forgot to mention one step.

Before suction of old medium in old cell dish, I treat 0% m199 to new cell dishes

(Dishes that huvec cells from old dish will be seeded) and suck up with aspirating pipet

anchored with pipet tip at the suctioning part

for washing process.

I use same aspirating pipet tip in later steps and

I also do not change aspirating pipet tip during other huvec works.

So if contaminations have not occurred, no problem with all of my huvec works, right? And which other problems as well as contamination occur when

I scratch huvec cell layer during huvec works?

Sorry for additional questions, but I am an unexperienced and young researcher

So I want you to understand my too basic and repeated questions.

Thank you!

huvecs are quite easy to come off. I use to wash cells twice with PBS and then add some trypsin quicky and detach cells mechanically with pipetaid but after neutralisation with M199 with SVF I spin the cell to remove all the trypsin. If you use filtered tips you should not get nay contamination.