Jc 1 probe

The mitochondrial transmembrane potential (Δψm) was assessed with the JC-1 probe (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzamidazolylcarbocyanine iodide, Thermo Fisher Scientific). This lipophilic membrane-permeant cation exhibit potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from ∼525 nm (monomeric form) to∼590 nm (aggregated form). Monocytes were seeded into 6-well plates (10⁶ cells/well) in medium with 10% human serum and incubated until semi-confluence. The medium was then replaced by CM in treated wells or by the medium in controls. After 24 h incubation, cells were stimulated with 1 μg/ml of LPS (Sigma-Aldrich) for 30 min. Cells were trypsinized, resuspended in 1 ml of PBS, and incubated with 10 μg/ml of JC-1 dye for 10 min at 37°C and 5% CO₂. Then, cells were washed and resuspended in PBS. Both red and green fluorescence emissions were analyzed by flow cytometry using an excitation wavelength of 488 nm and observation wavelengths of 530 nm for green fluorescence and 585 nm for red fluorescence, in a Becton Dickinson FACS-Canto II cytometer (BD Biosciences).

In a 24-well plate, 5 × 10⁴ cells/well were plated in DMEM/F12 10% FBS. In the next step, the neutrophil purification was started, and the groups were prepared as previously described. After polarization, neutrophils were centrifuged at 1000× g for 10 min, the supernatant discarded, and the cells resuspended in RPMI-1640 10% FBS. The culture medium was removed from all wells of the plate containing MDA-MB-231, and a 0.4 µm transwell was allocated to all wells. Neutrophils (5 × 10⁵ cells/insert) were placed on top of the inserts. After 20 h of stimulation, the inserts with neutrophils were discarded, as well as the medium remaining in the plate, and the tumor cells’ mitochondrial transmembrane potential was assessed in EnVision™ using JC-1 probe (10 μg/mL, Thermo Fisher Scientific), according to the manufacturer’s instructions.

Analyses of autophagy, apoptosis, mitochondrial membrane potential and proteasome peptidase activity were performed on a BD Accuri C6 flow cytometer (BD Biosciences). Annexin V-FITC/PI apoptosis detection kit was used as described by the manufacturer (Thermo Fisher Scientific). MAP1LC3A activation was measured using the FlowCellect Autophagy LC3 Antibody-based Assay Kit (Merck Millipore). Mitochondrial inner membrane potential was measured with the JC-1 probe (Thermo Fisher Scientific) as previously described (Agier et al., 2012 (link)). The peptidase activity of proteasomes was monitored using the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin, LLVY-AMC (Sigma-Aldrich) (Bulteau et al., 2006 (link)).

Cells were cultured for 2 days on glass coverslips coated with poly-l-lysine. A TCS SP2 AOBS confocal microscope (Leica Microsystems, Mannheim, Germany) was used with a PL APO 100 × /1.4–0.7 oil immersion objective (pinhole, 1 Airy unit) and a thermostable sample chamber (37°C) supplied with 5% CO₂ and either atmospheric air or humidified N₂ and 5% O₂. Note that this parameter is an ambient value, since the effective oxygen tension established at the cell layer seeded onto bottom of a culture flask is lower. Monitoring of mitochondrial membrane potential Δψ_m was done using the ratiometric JC-1 probe (Thermo Fisher Scientific) [see Supplementary material in JeŽek et al. (51 (link))]. Excitation was set at 488 nm, and emissions were collected between 500 and 600 nm under a confocal microscope. Ratio of JC-1 fluorescence at 593 vs. 537 nm has been shown previously to fall into a range of 0.2 to 0.4 in our experimental setup, the former value corresponding to the lowest potential near zero and the latter value to the high Δψ_m.