Alexa fluor 488 conjugated anti rabbit secondary antibody

After coculturing with MDA-MB-231 cells in the Transwell insert and microfluidic device, MCF7 cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were then washed three times with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 min at 4°C. The samples were washed three times with PBS, and nonspecific binding sites were blocked with 5% bovine serum albumin in PBS for 1 h at room temperature. Rabbit anti-GLUT1 (1:100; Abcam, Cat# ab15309, RRID: AB_301844), anti-PKM2 (1:100; Cell Signaling Technology, Cat# D78A4, RRID: AB_1904096), and anti-phospho-PKM2 (S37) (1:100; Thermo Fisher Scientific, Cat# PA5-78107) antibodies were diluted in PBS and incubated with the samples at 4°C overnight. In the microfluidic system, the diluted primary antibodies were added to the microchannels and incubated at 4°C overnight. The samples were then washed three times with PBS and incubated with an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:10,000; Invitrogen, Cat# A32731, RRID: AB_2633280) for 1 h at room temperature. Cell nuclei were stained using VECTASHIELD^® mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories). Fluorescent images were acquired using a confocal laser scanning microscope (Leica TCS STED CW).

Cells (4 x10⁴) grown in 8-well chamber slides were transfected with indicated siRNA for 48 h prior to mock infection or infection with SeV for additional 16 h. Cells were fixed with 3.7% formaldehyde, permeabilized in 0.2% Triton X-100, and subsequently immunostained with rabbit anti-IRF3 pAb at 1:400 or mouse anti-EAP30 mAb (1:100) followed by Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Invitrogen) or Texas Red-conjugated anti-mouse secondary antibody (Southern Biotech) at 1:200. After counterstaining the nuclei with 4', 6-diamidino-2-phenylindole (DAPI), slides were mounted and examined by fluorescence microscopy.

All buffers and solutions used were filter-sterilized, and samples were washed in 0.2% (v:v) Tween-20 in PBS, including two centrifugation steps (3700 g, 10 min, 4 °C). Samples were blocked (5% w:v BSA/2% v:v goat serum) for 30 min, washed, and then aliquoted for staining.
For FACS Aria cell sorting and ImageStream image cytometry, samples were aliquoted to 3 groups: an unstained control, an IgG isotype control (Abcam; diluted 1:100; 2 μg/mL), and an anti-human IgG stained sample (Abcam; diluted 1:150; 3 μg/mL). Aliquots were washed and incubated with AlexaFluor 488-conjugated anti-rabbit secondary antibody (Invitrogen; diluted 1:300; 7 μg/mL). To identify live bacterial cell populations, aliquots containing IgG isotype and IgG were stained with either propidium iodide (PI; Life Technologies; diluted 1:5; 50 μg/mL) 15 min prior to sorting FACS Aria samples, or with 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher; diluted 1:1000; 1 μg/mL) immediately prior to image flow cytometry.