5 µl aliquots of the PCR products with 1 µl loading buffer were analyzed by electrophoresis using a 1% (w/v) agarose gel in Tris Boric acid EDTA (TBE. 1X) buffer at 100 V for 45 min.
After washing with PBS buffer, cells were analyzed for mean fluorescence intensity (MFI) using a guava easyCyte HT instrument based on the manufacturer's instructions (Millipore, Danvers, MA, USA).
After washing with PBS buffer, the cells were analyzed for fluorescence intensity and human isotype IgG1 antibody was used as the reference sample control.
Total cell extracts were prepared with cell lysate buffer, and cell extracts were analyzed for protein content by SDS-PAGE as described previously [ 21].
PFAA-free hay, PFAA hay, concentrate, distilled water used for buffer preparation and nylon bags were analyzed for contents of PFAAs.
The cells were incubated on ice for 1 h, washed with cold PBS once again, and re-suspended in 300 μL of 1× annexin-binding buffer, and the stained cells were analyzed for apoptosis by flow cytometry soon after staining.
Luciferase expression was determined using a luciferase reporter assay; 1 × 10 cells were lysed in 100 μl Passive Lysis Buffer (Promega), and the lysates were analyzed for luminescence with the use of the Dual-luciferase Reporter Assay System (Promega, Leiden, the Netherlands) according to manufacturer's protocol and a Victor 3 luminometer (PerkinElmer, Groningen, the Netherlands).
After respiration assay, media were aspirated, wells were washed once with PBS, 25 μl of M-Per lysis buffer (Thermo Scientific) was added, and lysates were analyzed for total protein content using BCA assay (Pierce, Thermo Scientific).
After washing twice with washing buffer, DAPI was added and CD8+ T cells were analyzed for CD107a expression.
