The application protocol was developed to isolate high yields of viable endothelial cells from mouse neonatal brain tissue. Cells can be cultured or analyzed by flow cytometry afterwards.
PEB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091-222). Keep buffer cold (2−8 °C). Always use freshly prepared buffer. Do not use autoMACS Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as mouse or rat serum albumin, mouse or rat serum, or FBS. Buffers or media containing Ca2+ or Mg2+ are not recommended for use.
Coat the culture dish with 100 μg/mL fibronectin (overnight).
▲ MicroBeads concentrations below are optimized for the processing of 10⁷ total cells from neonatal mouse brain.
▲ For details on the use of the gentleMACS™ Dissociators, refer to the gentleMACS Dissociator user manuals.
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▲Volumes for magnetic labeling given below are for up to 1×10⁷ total cells. When working with fewer than 1×10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁷ total cells, use twice the volume of all indicated reagent volumes and total volumes).
▲For optimal performance it is important to obtain a single‑cell suspension before magnetic labeling. Pass cells through 70 μm nylon mesh (Pre-Separation Filters (70 μm), # 130-095-823) to remove cell clumps which may clog the column. Moisten filter with buffer before use.
▲Always wait until the column reservoir is empty before proceeding to the next step.
▲The recommended incubation temperature is 2–8 °C. Higher temperatures and/or longer incubation times may lead to non‑specific cell labeling. Working on ice may require increased incubation times.
▲ For a detailed immunofluorescent staining protocol, refer to the data sheet of CD31 Antibodies, anti-mouse.
▲ Note: Make sure the antigen epitope that is necessary for downstream applications is preserved during the dissociation procedure.
For a detailed list of antigen compatibilities and the right choice of Neural Tissue Dissociation Kit (NTDK) refer to the table on the product page at www.miltenyibiotec.com.
In case your epitope of interest is not listed please contact technical support. You can also perform a staining experiment with this antibody after using different enzyme concentrations, i.e., different dilutions of Enzyme P or T (e.g., for NTDK (P) 1:5, 1:10; for NTDK (T) 1:2.5) prior to isolation experiments to analyze the stability of your antibody epitope.
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