DME/F12 – Mammary Epithelial Growth Media 11/00 Make a 1:1 mixture of DME and F12 media Add these supplements to a 500 ml bottle of DME/F12; 1 tube of each. Final conc. 1. hEGF 0.5 ml of 10 µg/ml 10 ng/ml 2. Hydrocortisone 0.5 ml of 0.5 mg/ml 0.5 µg/ml 3. Insulin […]
Stock concentration Storage 1 Neomycin (G418) 500 mg/ml -20oC 2 Puromycin 2.0 mg/ml -20oC 3 Hygromycin 100 mg/ml 4oC T.C. 4 Zeocin 100 mg/ml -20oC 5 Blasticidin 10 mg/ml -20oC Information on ordering and aliquoting : Neomycin (G418, Geneticin) – Life Tech. 11811-031 5g $265.90. Comes lyophilized, store at RT. Dissolve in PBS-. […]
Adapted by JAR 10/4/09 ELISA PROTOCOL ( Click to Download ) ELISA kit: cat # _____________ from EBioscience Reagents: Buffers: 1. Coating Buffer 1 packet ELISA coating buffer (cat # 00-0044-59) 1L DI water filter using a 0.22 µm filter 2. Wash Buffer 1X PBS + 0.05% Tween 3. Assay Diluent 10 ml 5X assay […]
Horizontal Spread Assay (on 10T1/2 mouse fibroblasts) 10T1/2 fibroblasts work well for this assay because they grow well in DME 10% IFS, are infectable with high efficiency, and can be killed off rather quickly with standard drugs. Also, for routine passaging, the cells can also be split sparsely (a 1:10 or 1:15 split is usually […]
1) Freshly dissociate organoids into single cells and then seed 500K in 500µl/well in a 48 well non-adherent plate with protamine sulfate with virus at MOI 3 for infection overnight. (Patty and I have both stopped spin infecting the cells. This method seems to produce less cell death. I need to infect roughly 2x the […]
