I use 150k cells per well and these cells were plated after transfection. It's not due to the transfection as cells recover just fine when in 6-well plates. But everytime I work with smaller plates like 24 wells cells don't attach or survive. Also, when I do colony picking into 48- and 96-well plates they also do not survive. How do I solve the problem of hESCs not being able to attach and grow in plates with smaller diameters?
Hi Dee, I had problems with 24 well plates because the cells tended to aggregate in the middle of the well and die.
I found that it helped to add more media to the wells than recommended so that the meniscus was not close to the well bottom. Also after seeding the plates, gently move the plate in an x and y directions to disperse the cells.
If your incubator is not balanced properly, then calls can aggregate to a specific point on the plate, but you can continue swishing the plate until you are satisfied that they at properly dispersed.
Amended on 30 April 2018
I must sadly and repeatedly say that cultured cell line already has invaded microbes (please see file; HepG2 fucoidan).
Fetal hepatocyte Hc cells have
Aerobic；Arabidopsis thaliana (thale cress), Bacillus subtilis, Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa PA7, Caulobacter crescentus, Helicobacter pylori J99, and Lesionella pneumophila.
Anaerobic；Yersinia enterocolitica, Streptococcus pyogenes, Streptococcus mutans, Mycoplasma agalactiae, Mycoplasma capricolum, and Treponeme pallidum.
at 14.0% among cell proteins, and virus at 5.0% among cell proteins.
hESC seems to be recently established cell line, and may have many bacteria. Anaerobic condition is profitable to Anaerobic bacteria. Then, changes in aerobic condition seem to induce cell death in newly established cell lines.
Therefore, addition of Erythromycin in the culture medium is recommended.
Hepatoma HepG2 cells (established at 43 years ago (from 15y, male, Caucasoid) and antibiotics may have been thoroughly effected) have virus at 7.2% among cell proteins without bacteria. HepG2 cells show no growth changes by aerobic or anaerobic conditions.
Dr. Steingrimur Stefansson is only threatening the new researcher. Important point is how to rescue the sad situation of Dr. Dee Aa.
By the way, Dr. Masayoshi Namnba (Hon. Prof. of the Okayama University, School of Medicine, Okayama-city, Okayama, Japan) has independently established hepatoma cell line HuH-7 (from 57y, male, Japanese or Mongoloid) at 36 years ago.
I have previously reported about HuH-7, HuH-6, HepG2, and rat RLC-16 in 13 years ago (please see file; D-Asp Avidin 1). In this report, important findings are reported; i.e., (1) D-aspartic acid (10 mM) has induced cell death in rat RLC-16, and (2) Fucan (derived from Futo-Mozuku of Okinawa, Japan) has induced cell death in Hepatoblastoma HuH-6, but no effect to other adult Hepatoma cell lines of HuH-7 and HepG2.
Therefore, edible Fucan is effective to children's cancer, but no effect on adults' cancer. Reverse conclusion has been found that edible Fucoidan (sulphated poly-L-fucose) is effective to adults' cancer, but no effect to children's cancer and fetal hepatocyte Hc cells (please see file; Rat DEN Np-Fuco).
Hi Kou, Dee is working with hESC and they are fine in 6-well TC dishes.
If there was an infection it would not matter how the cells were grown. I have ~20 years of hands on experience with growing cells in tissue culture and I can tell you that when you have a bacterial infection, you will see it immediately.
Your posted paper has nothing to do with hESCs and is totally irrelevant to this topic.
Just curious, how long have you personally spent in the lab growing cells by yourself without the help of a team of technicians?
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