Alexafluor 488 conjugated polyclonal donkey anti mouse igg

Flow cytometric analysis was performed immediately after cell isolation as well as after 1, 4 and 7 days in culture. The antibodies used were anti α-actin monoclonal mouse IgM (Santa Cruz Biotechnologies, Santa Cruz, CA, cat. no. sc-58670), anti β-actin monoclonal mouse IgG1 (Invitrogen, Carlsbad, CA, cat. no. AM4302), AlexaFluor488-conjugated polyclonal donkey anti-mouse IgG (Invitrogen, cat. no. A-21202), DyLight594-conjugated polyclonal goat anti-mouse IgM (Abcam, Cambridge, MA, cat. no. ab97009) and FITC-conjugated monoclonal anti-CD117 mouse IgG (Millipore, Temecula, CA, cat. no. MAB1162F). Briefly, 5×10⁵ cells were collected and washed in flow buffer (PBS + 10% FBS + 1% Sodium azide). Direct extracellular staining of CD117 (c-Kit) was performed using the appropriate dilution of fluorescence conjugated primary antibody in flow buffer. Cells were then fixed in 100% methanol and incubated in permeabilization buffer (PBS + 0.5% Triton X-100). For intracellular indirect co-staining cells were stained with the primary antibody in blocking solution (PBS + 0.1% BSA + 0.2% Triton X-100 + 0.05% Tween-20 + 10% goat serum) and washed in PBS/T (PBS + 0.1% Triton) before incubated in the appropriate dilution of fluorescent secondary antibody. Analysis was performed by a FACSCalibur flow cytometer (BD Bioscience, San Diego, CA) and FlowJo software (Tree Star, Ashland, OR).