The adherent cells were washed three times with phosphate buffered saline (138 mM NaCl; 2.7 mM KCl; 1.5 mM KH2PO4; 10 mM NaH2PO4, pH 7.5), then overlaid with 400 μL of Lysis Buffer (1% Triton X-100, pH 7.5, 20 mM HEPES buffer with 0.1 mM EDTA), scraped and collected into a glass vial for tocopherol analysis by HPLC.
IgG bound to the column was eluted using acid glycine (0.1 M glycine, pH 2.0) and collected into neutralization buffer (1 M unbuffered Tris).
Before use in flow cytometry, MФs were infected as previously described, washed five times with PBS for 5 min each, and collected into 1× Extraction Buffer with protease inhibitor cocktail (Sigma Aldrich).
Captured DNA was finally eluted with 5 20 μL of elution buffer (10 mM Tris-HCl, pH 8.5) and collected into a microcentrifuge tube.
Cells were washed twice with ice-cold PBS and collected into 200 µl homogenization buffer (20 mM Na-HEPES, 1 mM EDTA, 0.32 M sucrose, pH 7.0).
Tumour sites and normal lungs in FFPE tissue sections were dissected and collected into individual tubes filled with PKD buffer of the RNeasy FFPE kit (Qiagen, Venlo, Netherlands).
The rehydrated tumor tissue was microdissected and collected into a centrifuge vial with 25 μl of PBS buffer.
Sterile phosphate buffered saline at 37°C was instilled in five aliquots of 50 ml and immediately re-aspirated and collected into a siliconised plastic bottle kept on ice.
Next, the pellet containing the plasma membrane fraction was suspended in SHB buffer and collected for Western blot analysis.
Approximately 500 grams of L. minor fronds were crushed in pestle and mortar with 5 ml of potassium phosphate buffer, collected into 15 ml centrifuge tube and centrifuged at 12,000 rpm for 20 min.
