Isolation and Quantification of Murine Macrophages

Healthy murine mammary tissues and EO771 tumors were processed to generate tissue homogenates via mechanical disruption in the presence of Liberase TM (Millipore Sigma) per manufacturer’s protocols. Macrophages from healthy spleen and mammary tissues or EO771 tumors were isolated via flow-sorting sized, LiveDead negative, CD45⁺CD11b⁺F4/80⁺ cells using a MoFlo Astrios High Speed Sorter (Beckman Coulter). Subsequently, cells were lysed in TRIzol reagent (Invitrogen) and RNA was precipitated according to manufacturer recommendations. The isolated RNA was reverse transcribed with SuperScript (Invitrogen). The product of the reverse transcription was then used in triplicates for a qRT-PCR reaction with SYBR Green Fast (ABI Inc.). The CT averages were calculated for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NRP2a, and NRP2b primers as reported previously (8 (link)). Results are reported as the percentage of GAPDH using the formula: %GAPDH = (2−ΔCt) × 100.