善本生物

ALIASES

ACSS2; Acyl-CoA Synthetase Short Chain Family Member 2; ACS; DJ1161H23.1; ACAS2; ACSA; Acetyl-Coenzyme A Synthetase 2 (ADP Forming); Acetyl-Coenzyme A Synthetase, Cytoplasmic; Acetyl-CoA Synthetase 1; Acyl-Activating Enzyme; Propionate--CoA Ligase; EC 6.2.1.1; AceCS1; AceCS; ACECS; Acyl-CoA Synthetase Short-Chain Family Member 2; Cytoplasmic Acetyl-Coenzyme A Synthetase; Acetyl-CoA Synthetase; Acetate--CoA Ligase; Acetate Thiokinase; Acetate-CoA Ligase; EC 6.2.1.17; EC 6.2.1

Background

NCBI Gene Entry: 55902

TESTED CELL LINE

HT-1080

VALIDATION METHODS

RT-qPCR; Western Blotting (WB)

SHIPPING

Shipped with dry ice. Immediately store the product in a standard freezer at -80°C upon receipt.

STORAGE

1. Lentiviral particles, -80 °C for 1 year. 2. Cell lysate kit, -20 °C for 2 years. 3. Antibody, -20 °C for 1 year.

INSTRUCTIONS FOR USE

The following protocol uses HT-1080 cell as an example assuming your cell culture medium is DMEM-based. 
1.Release 0.5 million HT-1080 cells into a 35 mm tissue culture dish in 2 mL of the growth medium (DMEM containing 10% FBS and 1% pen/strep). Cell density should reach 50-60% confluence the following day.
2.24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
3.Discard 1 mL of the original growth medium of the 35 mm dish.
4.Using a serological pipette, gently mix the lentiviral solution 3 times.
5.Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
6.Add a polybrene stock solution to the culture medium at a final concentration of 5 μg/mL. Gently swirl the dish to mix.
7.48 h after cell release, without discarding the original medium, add another 1 mL of lentiviral medium directly into the dish.
8.Add an additional polybrene stock solution into the dish to obtain a final concentration of 5 μg/mL. Tip: Now, the medium in the dish should be a total of 3 mL.
9.72 h after cell release, cells may reach confluence. Trypsinize the cells off the 35 mm dish and culture those cells in a 60 mm dish.
10.Add puromycin to the dish at a final concentration of 4 μg/mL. Tip: To assess the efficacy of puromycin selection, culture a dish of wild-type HT-1080 cells as a negative control.
11.Allow puromycin selection for 48 h. Almost all wild-type HT-1080 cells should die, while the dish infected with lentiviruses should have some remaining cells.
12.Replace the medium with regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.

Note: 1. This product is for research use only. 

      2. This product is only supplied to end users.

      3. Do not use this product for commercial.