pLV-EF1a-IRES-Puro

Protocol tips

Upstream tips	Protocol tips	Downstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

KO cell lines and cell culture
Rip1 KO, Rip3 KO and Mlkl KO L929 were described previously17,19. Ppm1b, Ppm1a, TNFRI and IKKβ KO cell lines were generated using the CRISPR/Cas9 technology30,49. Rip1–Rip3 DKO L929 cells were generated by targeting Rip1 in Rip3 KO L929 cells. The targeting sequences are: Ppm1b-5′-CTGGGAATGGTCTGCGTTA-3′; Ppm1a-5′-TGTAATCGAAATCCCCAA −3′; Rip1–5′-AACCGCGCTGAGTGAGTTGG-3′; TNFRI-5′-GCTTCAACGGC ACCGTGACA-3′; IKKβ-5′-CACCGTGACCGTTGACTAC-3′. Thioglycollateelicited peritoneal macrophages were obtained from eight-week-old WT or Rip3 KO mice using a method described previously50. Primary WT and Ppm1bd/d MEF cells were generated from day 14 embryos of the same litter of Ppm1b+/d heterozygous breeding. The primary MEF cells were immortalized by infection with retrovirus encoding E1A. Mouse BMDMs were differentiated in vitro from isolated bone marrow cells. Bone marrow cells collected from mouse femurs and tibias were incubated for seven days in DMEM containing 20% heat-inactivated FBS, penicillin, streptomycin and 30%L929 conditional medium28. Three pairs of Ppm1bd/d and WT mice were used to produce MEF cells and BMDM cells in this work, respectively. HT-29 cells were cultured in McCoy’s 5A culture medium (Invitrogen); L929, HEK-293T, NIH3T3-A, NIH3T3-N, HeLa, peritoneal macrophage, J774 and MEF cells were cultured in DMEM. All media were supplemented with 10% FBS (vol/vol), 2 mM L-glutamine, 100 IU penicillin and 100 mg ml−1 streptomycin at 37 °C in a humidified incubator containing 5% CO2.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pLV-EF1a-IRES-Puro below.

Download manufacturer protocol

Videos

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