i have tried 0.5-1 ug/ml on my NSCs with puromycin resistance... and it worked fine.

good luck!

Before starting selection of transduced clones, a wide range of PURO should be applied to optimize the concentration as you did. However, 10ug/ml is very high concentraiton for NCs. While fibroblast cells might be very resistant to high PURO concentrations (eg. 15 ug/ml in our lab), neural cells would be very sensitive. I suggest you to try several concentrations between 0.01-2 ug/ml to find the best concentration for selection. 

We use human bone marrow derived MSC and we have noticed that these cells are relatively sensitive to Puromycin even at 1ug/ml and they are also donor dependent. As suggested by Selami Demirci  a lower titre of Puromycin is needed, and also if you are using NCS from different donors, perform the titre on these finding the optimal con for all you lines.

Good Luck

Hello

We used HESC and followed this Protocol :

Prior to transduction — establish the Puromycin Kill Curve

Determine the minimal puromycin concentration to kill all non-transduced cells. Use a range starting from 1–10 µg/ml of puromycin on the cells.

Transduction Procedure

DAY 0

0.1 Passage cells when 80% confluent with large colonies approximately 24 hr prior to initial transduction using dispase at 1 mg/ml.

0.2 Seed hESC cultures at a high density into a 24-well culture dish, taking care to maintain cells as aggregates of approximately 50–60 µm in diameter.

DAY 1 (late afternoon)

1.1 Feed hESC cells with 500 µl of pre-warmed (37 °C) mTeSR1 containing 6 µg/ml of polybrene (Hexadimethrine bromide).

1.2 Replace cultures into incubator for 15 min.

1.3 Add the desired amount of virus particles to the culture medium.

10 µl of 1x106 TU/ml viral particles for H1 and H9 hESC lines. Since an exact cell count cannot be determined from the previous day’s plating due to extreme aggregation of the cells (ranging from 50 to 200 cells per clump), a precise MOI cannot be calculated. The most effective amount of virus particles to be added should be determined empirically for each cell line.

1.4 Incubate for 18–20 hr at 37 °C, with 5% CO2 and 95% humidity.

DAY 2 (mid-day)

2.1 Remove medium, and replace with 500 µl of pre-warmed media supplemented with 6 µg/ml of polybrene.

2.2 Replace cultures into incubator for 15 min.

2.3 Add three times the initial amount (from Day 1) of viral particles to the culture medium.

2.4 Incubate for 18–20 hrs at 37 °C, with 5% CO2 and 95% humidity.

DAY 3 and 4 (morning)

Remove medium daily and replace with 500 ml pre-warmed media without polybrene.

DAY 5–8

Remove media daily and replace with 500 µl pre-warmed media supplemented with puromycin. Again, a puromycin kill curve should be run prior to any transduction experiment requiring puromycin selection.

Not Dr. Cohen’s lab determined that a final concentration of 1 µg/ml puromycin worked best to select for successfully transduced H1 and H9 hESCs.

DAY 9+

To determine the efficacy of shRNA-mediated knockdown, carry out molecular assays to quantify mRNA and protein levels. Be sure to compare to appropriate controls.

For subsequent analysis of additional time points, continue culturing hESCs with puromycin-supplemented media.

Good Luck