Alexa fluor 488 secondary antibody a 21206

PAMs were seeded at a concentration of 1.5·10⁶ cells/well and mock-infected or infected with either Arm07/CBM/c2 (WT) or Arm/07-ΔMGF505-2R-GFP at a multiplicity of infection (MOI) = 0.5, in DMEM–10% porcine serum. Cells were then collected at 0, 24, 48, or 72 hpi and subjected to freeze–thaw cycles (×3). For quantification, titration was performed by a Reed & Muench TCID-50 assay [44 (link)] in COS-1 cells. Briefly, several dilutions of each sample were used to infect COS-1 cells s (7000 cells/well) and 72 hpi. They were fixed with PFA 4%, and permeabilized with 0.2% Triton X-100 (in the case of Arm/07/CBM/c2). TCID50 titration assay was performed by staining viral p32 (with anti-p32 primary antibody (S-5C1) (1/500) incubation followed by anti-mouse Alexa Fluor 488 secondary antibody (A-21206) incubation, from Invitrogen) for Arm/07/CBM/c2or using green (GFP) fluorescence (in the case of Arm/07-ΔMGF505-2R-GFP). Biological duplicates were used.

Porcine alveolar macrophages (PAMs) were seeded in M12 plates (1.5 × 10⁶ cells/well) and mock or infected with Arm07/CBM/c2 (WT) or Arm-ΔCD2v-ΔA238L (0.5 PFU/cell) in DMEM–10% porcine serum. At 0, 24, 48 or 72 h post-infection (hpi) the cells were collected and subjected to three freeze–thaw cycles. Total virus was titrated in COS-1 cells by using a TCID-50 assay. Briefly, COS-1 cells were seeded in p96 plates (7000 cells/well) and infected with several dilutions of each sample. After 72 hpi cells were fixed (PFA 4%), and in the case of Arm/07/CBM/c2, permeabilized with 0.2% Triton X-100, and a TCID50 titration assay was performed using green (GFP) and red (mCherry) fluorescence for Arm-ΔCD2v-ΔA238L or using viral p32 staining using anti-p32 primary antibody (S-5C1) (1/500) and anti-mouse Alexa Fluor 488 secondary antibody (A-21206) from Invitrogen for Arm/07/CBM/c2. Biological duplicates were used.