| Product Name |
Human DPYD Knockdown Cell Line (WB-Validated) |
| Aliases |
DPYD; Dihydropyrimidine Dehydrogenase; DPD; Dihydrothymine Dehydrogenase; Dihydrouracil Dehydrogenase ; Dihydropyrimidine Dehydrogenase [NADP(+)]; EC 1.3.1.2; DHPDHase; DHPDHASE; DYPD; DHP |
| Background |
Gene Name: DPYD
NCBI Gene Entry:
1806
|
| Storage |
Cell Line: Store at liquid nitrogen for 1 year.
Antibody: Store at -20 °C for 1 year. |
| Kit Components |
1. Human DPYD Knockdown Cell Line (WB-Validated) (1 mL, 1×10⁶ cells )
2. Wild-type cell line (1 mL, 1×10⁶ cells )
3. Verification Tool: KD-Validated Dihydropyrimidine Dehydrogenase Recombinant Rabbit mAb #61151 (5 μL) |
| Parental Cell Line |
Human cell line supplied by the client |
| Validation Methods |
RT-qPCR, Western blotting (WB) |
| Shipping |
Cell Line: Shipped with dry ice. Immediately store the product in liquid nitrogen upon receipt.
Antibody: Shipped with ice packs. Immediately store the product at -20°C upon receipt. |
| Instructions For Use |
This knockdown cell line should be paired with wild-type cell line for use. |
| Manufacturing Process |
The following protocol was used to generate mRNA knockdown cells:
1.Release 0.5 million 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 cells as a negative control.
11.Allow puromycin selection for 48 h. Almost all wild-type 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 |
This product is for research use only. |
