Bovine serum albumin (bsa)

The choroid plexus was explanted 48 h post adoptive cell transfer from the fourth ventricle and transferred on glass object slides and incubated in PBS (PAN Biotech) + tween20 (0.3%; Sigma) at RT for 5 min, washed twice in PBS (PAN Biotech) for 5 min and fixed in PBS (PAN Biotech) + PFA (2,2%; Sigma), glucose (2%, Sigma), sodium acide (0.02%; Sigma) for 20 min at RT. Choroid plexus were then rinsed in PBS (PAN Biotech), fixed in methanol (100%, Sigma) for 6 min at RT, washed twice in PBS for 5 min and blocked in PBS (PAN Biotech) + BSA (1%; PAN Biotech), tween20 (0.3%; Sigma), normal goat serum (10%; Sigma) for 30 min at RT. The staining was performed in PBS (PAN Biotech) + tween20 (0.3%; Sigma) + primary antibody for 2h at RT (rat anti CD31; 1:100; BD). After washing the tissue twice in PBS (PAN Biotech) for 5 min, secondary stainings were performed using anti rat secondary antibodies (donkey anti rat-Alexa647; life technologies). Finally, the tissue was stained with DAPI (1 µg/ml; Sigma) and embedded in fluorescent mounting media (Dako).

The following buffers and chemicals were used: erythrocyte lysis buffer (Qiagen, Hilden, Germany), 1 × PBS, made from 10 × PBS (Rockland, Gilbertsville, PA, United States; pH 7.2) using Millipore water, 0.1% cell permeabilization buffer, made from 10 × saponin-based permeabilization buffer (eBioscience), 4% formaldehyde solution in PBS made from 16% paraformaldehyde (EMS, Hatfield, PA, United States), PBS supplemented with 0.5% BSA (PAN Biotech, Aidenbach, Germany), and 0.02% sodium azide (Sigma-Aldrich, St. Louis, MO, United States) (PBS/BSA). Buffers were sterile-filtered through 0.22-μm membranes and stored in Stericup disposable bottles (Merck, Darmstadt, Germany). Blood, urine, and cells were processed in 50-ml, 15-ml, and 5-ml round-bottom polystyrene/polypropylene tubes (Corning, Corning, NY, United States and Sarstedt, Nümbrecht, Germany).
MAXPAR antibody labeling kits, EQ Four element calibration beads, washing and tuning solution, and DNA intercalators were purchased from Fluidigm Corporation (South San Francisco, CA, United States). Pre-conjugated and unlabeled antibodies are summarized in the Supplementary Table S1.
Cis-Platinum (II)-diamine dichloride (cisplatin) was purchased from Enzo Life Sciences GmbH (Lörrach, Germany). A 25 mM stock solution was prepared in DMSO (Sigma-Aldrich) and aliquots were stored at -20°C.

Cells were fixed with 4% paraformaldehyde (PFA, ThermoFisher, cat. no. 28908) in 1X PBS for 10 min, and then, permeated with 0.1% Triton X-100 (Sigma-Aldrich, cat. no. T8787) in 1X PBS for 15 min and blocked with 5% bovine serum albumin (BSA, PAN-Biotech, cat. no. P06–139310) in 1X PBS-0.1% TWEEN20 (Sigma-Aldrich, cat. no. P2287) solution for 30 min. Samples were incubated with a primary antibody against NF-κBp65 (1:200, Abcam, cat. no. ab1652) overnight, followed by a secondary antibody (1:500, Invitrogen, cat. no. a11036) and Hoechst 33342 (1:1000, Invitrogen, cat. no. H3570) for 1 h in the dark. After three washes with 1X PBS, slides were observed under a Leica DMi8 microscope.

As described previously, six mM analytical grade fatty acids C16[1]c and C18[2]c (Biotrend Chemikalien GmbH, Cologne, Germany) were conjugated to 2.4 mM FA-free bovine serum albumin (BSA, PAN Biotech, Aidenbach, Germany) at a 1:2.5 ratio in water by incubation at 50 °C for 5 min [27 (link)]. An equivalent BSA solution was prepared without FAs for control experiments.

DEP buffer with low electrical conductivity was prepared to keep cells viable during the processes of dielectrophoresis. As it has been previously reported [31 (link)], the low conductive DEP buffer [48 (link)] was composed of 8.6% sucrose (product no: LC-4469.1, NeoFroxx, Hesse, Germany), 0.3% glucose (CAS number 59-99-7, Sigma-Aldrich, Darmstadt, Germany) and 0.1% bovine serum albumin in distilled water (BSA, product code: P06-1391050, PAN-Biotech, Aidenbach, Germany).
The conductivity of the DEP buffer was 0.002 S/m, as measured by a Corning Model 311 Portable conductivity meter at room temperature (Cambridge Scientific Products, Watertown, MA, USA).

Cells were fixed with 2% paraformaldehyde (PFA) w/o methanol (15 min, Thermo Fisher Scientific, Darmstadt, Germany), permeabilized, and blocked with 0.5% Triton X-100 (Thermo Fisher Scientific, Waltham, USA) in 2% BSA (PAN-Biotech, Aidenbach, Germany) for 60 min. ER stress markers included: Alexa 647 anti-ATF-4 antibody (B-3, 1:50, Santa Cruz), Alexa 594 anti-calnexin antibody (AF18, 1:50, Santa Cruz), and Alexa 488 anti-cytochrome c (1:50, Biolegend). For stemness, antibody mixtures, either containing anti-GFAP (1:200, Alexa Fluor 594, BioLegend, San Diego, USA) and anti-A2B5 (1:200, Alexa Flour 647, BioLegend, San Diego, USA), or anti-Oct-4 (1:500, Alexa Fluor 647, BioLegend, San Diego, USA) and anti-Nanog (1:500, Alexa Flour 488, BioLegend, San Diego, USA) were added and staining was done at 4 °C overnight. The next day, GFAP/A2B5-antibody mix was stained with Phalloidin green (1:50, BioLegend, San Diego, USA). Nuclei were counterstained with DAPI (1:1.000, Biomol, Hamburg, Germany) and cells were analyzed using a Zeiss microscope Axio Observer 7 (Zeiss, Oberkochen, Germany).