Cells do not attach in the flasks - (Oct/11/2008 )

Hi everyone!!!

I thawed my HepG2 cells (clones) (they were stored at -70 with DMSO) but i have faced a problem...... they can't adhere onto the flask.
They haven't looked dead, just they are floating on the medium.... it takes 6 days i thawed them!! I can't waste these cells because they are transfected with RNAi. i could notice increasing of cells but they can't adhere onto the flasks.
Medium is 199 with 20% SBF (used 10% before but trying to wake them up increasing the SBF), 1% antibiotics. Not using yet geneticin, to select my clones.
Incubator conditions are ok 5% CO2, 37ºC, clean.
Next, I certainly will store them in liquid Nitrogen!
any good suggestion???

thank you in advance

---

I am no expert. But this problems seems to have similarity to the post below.

http://www.protocol-online.org/forums/inde...showtopic=40058

---

hi thanks for replying!!!

i'm sure of, that the problem is not the flasks. i used the same in the past to work with these cells. this problem got when i thawed my clones (with iRNA) from -70. i use Corning flasks

any other advice??

Thanks again!

---

Some cell lines need coating prior to seeding. You may try fibronectin or collagen coating.

---

Hi Durandal!!
thanks for replying me!

Few months later i coiuld grow these cells easily with no coating ( i know that some cells need to collagen or fibronectin)
I stored these clones (they were transfected with iRNA) in -70º C for 10 months.
this strange problem started when i thawed them. They are not able to attach in the flasks, but i can see clearly their morphology (yes they are alive) and i can clearly notice to increase of amount cells.
What would yoiu advice me ??

Best!

Reis, V.P

---

Do you want to save the cells you have in suspension?

If so, spin them down, resuspend in higher serum and lower antibiotic. Plate on multiwell plates using the minimal amount to medium you can use (increase their contact to the chamber). Add your final medium at different intervals, you want to bring your antibiotic concentration up to selection soon, but not to soon.

Did someone thaw the -80ºC? How much serum/DMSO did you use in freezing?

Best of luck.

Alejandro

---

On the other hand.

Do you still have frozen aliquots? If so, time to break out a new batch. Try a modification of the previous protocol, but remember to dilute and spin. DMSO is poisonous to cells.

Good luck
A.

---

Hi alejandro!!

thank you for replying me!!

i've saved them and kept them alive spinning them down and changing the medium everyday but i use to ressuspend in the medium ( 199 with 20 % SBF). but unfortunatelly they still keep on floating..... some i can see attached. i'm sure that the cells floating on medium aren't dead. are there any reason or explanation for this??? i have frozen aliquots but i wouldn't like to waste cells (they are transfected with iRNA). i'll follow your advice and if it doesn't work........ i throw them out and start over again.

Best

Reis, V.P.

---

Just curious, what does the iRNA interfere with?

---

Hi Durandal!

Iwork at Virology Laboratory and we have been working with RNAi against Dengue Virus.
We made RNAi against differents regions of virus genome. we hope to inhibit virus replication on HepG2 cells, and vero cells.
My PHD (who guides me thru research, Yes i'm a student) is a very expert in RNA interference and molecular biology. If you want to keep in touch you may send me a private message.

Best

Reis V.P.

---