Pkh26 red fluorescent cell linker

The primary cortical culture was allowed to grow for 14 days after which they were infected with G1-NH₂, G4-NH₂, G1-90/10, and G4-90/10 dendrimers. These dendrimers were used at various concentrations incubated at 37 °C at three different time points to optimize the cellular uptake (data not shown). The final concentration of NH₂ surface dendrimer was optimized to be 0.5 mg/mL and mixed surface dendrimer was optimized to be 4 mg/mL, which were taken up by the cultured cortical culture cells (neurons and glial cells) in 30 min. The dendrimers were added to the cells after labeling the cells with PKH26 Red Fluorescent Cell Linker (Sigma Aldrich, St. Louis, MO, USA), according to the manufacturer’s protocol. The labeled culture cells were fixed with 4% paraformaldehyde (PFA), mounted, and viewed under Zeiss Observer inverted microscope, and confocal images were captured using Olympus BX50 Upright Microscope.

Splenocytes from naive mice were divided into two populations stained with carboxyfluorescein succinimidyl ester (CFSE - Molecular Probes) at a final concentration of 10 μM (CFSE^High) and 1 μM (CFSE^Low). PKH26 Red Fluorescent Cell Linker (Sigma-Aldrich) was also used at a final concentration of 20 μM. The target cells labeled with CFSE^High or PKH were pulsed with peptide VNHRFTLV or TEWETGQI for 40 minutes at 37°C according to each experiment’s concentration. CFSE^Low cells remained uncoated. Subsequently, all stained populations were counted and mixed at the same proportion. 4 x 10⁷ cells were transferred via intravenous into mice and after 14 hours, the spleens of recipient mice were collected to CFSE^Low, CFSE^High and PKH⁺ detection by flow cytometry using FACS Canto II. The percentage of specific lysis was determined by this formula:

A total of 100,000 cells of WT group marked with CytoTrace™ Green CMFDA (Thermo Fisher Scientific) according to the manufacturer’s instruction and 100,000 cells of the SAS group or FAS group marked with PKH26 Red Fluorescent Cell Linker (Sigma-Aldrich) were mixed and seeded on the 0.6 kPa PA gel and then incubated at 37 °C for 15 min. The cells were rinsed gently with PBS. The adhered cells remaining on the gels were counted.

The MLMs were organized with various cell types at different ratios: (i) HepG2 100%, (ii) HepG2 70%/LX2 30%, and (iii) HepG2 60%/LX2 20%/HUVEC 20%. Cell suspension (5,000 cells per well) was added to 96-well round-bottom ultralow attachment (ULA) plates [26 (link), 27 (link)]. The self-aggregation of the cells generated the microtissue structures after 4 days. Three different fluorescent dyes were used to follow each cell type in our complex model: PKH67 green fluorescent cell linker (Sigma–Aldrich, USA) for HUVEC, PKH26 red fluorescent cell linker (Sigma–Aldrich, USA) for LX2, and DAPI (4′,6-diamidino-2-phenylindole) as a blue-fluorescent dye for all cell nuclei (so HepG2 was determined by merging the figures). The diameter, circularity, and aspect ratio were determined by ImageJ software (http://imagej.nih.gov/ij/index.html).

For single cell type culture, MDA-MB-231 cells or CAFs were seeded at 8 × 10⁴ (time 0 h) per well on 6-well plates in Dulbecco’s Modified Eagle’s Medium (DMEM) without glucose, glutamine, and sodium pyruvate (Corning, Corning, NY, USA) that was supplemented with 3 g/L glucose, 10% fetal bovine serum and indicated levels of glutamine (0, 1, 2 or 4 mM) and ammonium chloride (NH₄Cl; 0, 5, 10 or 25 mM). At indicated time points (24, 48 and 72 h), cells were stained with trypan blue (to label dead cells), and live cell numbers were determined using a TC20 automated cell counter (Bio-Rad Laboratories, Hercules, CA, USA). For coculture, MDA-MB-231 cells labeled with PKH67 green fluorescent cell linker (Sigma-Aldrich, St. Louis, MO, USA) and CAFs labeled with PKH26 red fluorescent cell linker (Sigma-Aldrich, St. Louis, MO, USA) were mixed at 1:1 ratio, and a total of 8 × 10⁴ mixed cells were seeded. At indicated time points, numbers of each cell type were determined based on the different fluorescent labels.

Exosomes were labeled with PKH67 Green Fluorescent Cell Linker (Sigma Aldrich, St. Louis, MO) in a modified protocol as described previously [38 (link)]. A2780 cells were labeled with PKH26 Red Fluorescent Cell Linker (Sigma-Aldrich) according to manufacturer's instructions. Adhered A2780 cells were exposed to exosomes at a concentration of 1 μg/10,000 cells plated in time points of 0.5, 1, 2, and 24 hours. Media was removed upon completion of all time points and cells were gently washed once with PBS and fixed with 4% paraformaldehyde. After washing with PBS, cover slides were attached using a mounting medium containing DAPI. 3-6 images were taken of each time point and overlaid using MetaMorph Software (Molecular Devices, SunnyVale, CA).

Tumour masses were explanted and cells were disaggregated and labelled with PKH26 Red Fluorescent Cell Linker (Sigma Aldrich), according to the manufacturer’s instructions. Labelled cells were lysated, as previously described [17 (link)], and DCs were incubated with the labelled lysate for 24 hours. Cells were then harvested and stained with an anti-CD11c FITC monoclonal antibody for 24 hours at 4°C, fixed with paraformaldehyde 1% for 15 minutes, washed and mounted on slides using the Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Blue DAPI staining was performed to identify nuclei. Confocal fluorescent images were obtained using a LSM 510 Zeiss confocal scan head mounted on a Zeiss Axiovert 200 M on an inverted-based microscope using a 40x or 63x oil immersion objective. Sequential excitation at 488 nm and 543 nm was provided by argon and helium-neon gas lasers, respectively. Emission filters BP500-550 and LP560 were used for collecting green (FITC) and red (PKH26) in channels one and two, respectively. After sequential excitation, green and red fluorescent images of the same cell were saved with Laser Sharp software. Images were analysed by Zeiss software. The term co-localization refers to the coincidence of green and red fluorescence, as measured by the confocal microscope.