Fitc labeled goat anti mouse igg

KP bacterial cultures grown 12–18 hours at 37°C / 250 rpm in HS media were washed with PBS and the OD_600nm adjusted to 0.8 from which 100 μL were centrifuged 10 minutes at 4°C, 15,000 x g. The bacterial pellet was then suspended in 100 μL of mouse immune serum diluted in the flow cytometry staining buffer (1 x PBS, 1% fetal bovine serum [Gemini Bioproducts, CA], 0.02% sodium azide). The samples were incubated for 1 hour at 4°C, then washed three times with the staining buffer followed by incubation for 1 hour at 4°C with FITC labeled goat anti-mouse IgG (BioLegend, CA) at 9 ng/μL. Stained bacteria samples were acquired on a LSR-II (custom built) system (BD, San Jose, CA). FSC and SSC were set to logarithmic amplification. The data were analyzed using the software package FlowJo V10 (Ashland, OR).

Cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences), and intracellularly stained with mouse monoclonal anti-dsDNA (35I9 DNA, Cat#ab27156; Abcam; 1:600), anti-connexin 43 (F-7, Cat#sc-271837; Santa Cruz; 1:100) antibodies, or rabbit polyclonal antibodies against influenza virus M2 (1:600) or EMCV 2B (1:600) proteins followed by FITC-labeled goat anti-mouse IgG (Cat#405305; BioLegend; 1:300), PE-labeled goat anti-mouse IgG (Cat#405307; BioLegend; 1:300), or FITC-labeled donkey anti-rabbit IgG (Cat#406403; BioLegend; 1:300). Flow cytometric analysis was performed with a FACSVerse flow cytometer (BD Biosciences). The final analysis and graphical output were performed using FlowJo software (Tree Star, Inc.). All samples were gated based on forward scatter (FSC) and side scatter (SSC) to gate out cellular debris or dead cells.