Propidium iodide dye

Namalwa and Raji B cells were lysed in 1 mL RIPA buffer, and iron concentration in lysates was determined using the QuantiChrom^TM Iron Assay Kit (Bioassay Systems, Hayward, CA, United States). The number of live/dead cells was determined by flow cytometry using propidium iodide dye (Invitrogen).

The antibodies used in this experiment, including primary (Bak, Bax, Bcl-2, PARP, cleaved caspase 3, cleaved caspase 9, E-cadherin, N-cadherin, DVL-2, DVL-3, c-Myc, Cyclin B1, β-catenin, Histone 3 and β-actin) and secondary (secondary anti-mouse, secondary anti-mouse) antibodies, and anti-rabbit IgG (R) Alexa Fluor® 488 molecular probe and Anti-mouse IgG (R) Alexa Fluor® 488 molecular probe were purchased from Cell Signaling Technology (Danvers, MA, USA). Propidium iodide (PI) dye was purchased Life Technologies (Eugene, Oregon, USA). Fluoroshield mounting medium, containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), was purchased from Abcam (Cambridge, MA, USA). Crystal violet dye was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM) was purchased from GE Healthcare Life Sciences and HyClone Laboratories (Logan, Utah, USA). Green cytotox, Annexin red and caspase 3/7 IncuCyte reagents were purchased from Essen BioScience (Ann Arbor, MI 48108, USA). The 6.5 mm diameter inserts, with 8.0 μM pore size polycarbonate membrane for 24 well plates, were purchased from Corning Incorporated (One Riverfront Plaza Corning, NY 14831, USA). Growth-factor-reduced matrigel was purchased from BD Biosciences (Ontario, Canada).

Primary and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Propidium iodide (PI) dye was purchased from Life Technologies (Eugene, Oregon, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GE Healthcare Life Sciences, HyClone Laboratories (Logan, UT, USA). The 0.25% trypsin + 2.2 mM ethylenediaminetraacetic acid (EDTA) and phosphate-buffered saline (PBS) were purchased from Mediatech, Inc. (Corning subsidiaries, Manassas, VA, USA). Crystal violet dye powder was purchased from Sigma-Aldrich (St. Louis, MO, USA). Corning^® transwell inserts (24 mm Transwell with 8.0-μm pore polycarbonate membrane inserts, TC-treated, w/lid, sterile, 24/cs) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The compound 3-(4,5dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) was purchased from Calbiochem EMD Millipore (Billerica, MA, USA). Clarity™ and Clarity Max™ Western ECL blotting substrates were purchased from Bio-Rad Laboratories (Hercules, CA, USA).

hOLs viability was assessed by live staining using propidium iodide dye (PI; ThermoFisher Scientific). The average number of PI⁺ (dead cells) and total cell number (Hoechst) per condition was determined via Leica TSC SP8 confocal microscope and ImageJ. For apoptosis assay, cells were incubated in the presence of Cell Event Caspase 3/7 detection reagent (C10423, Invitrogen) for 1 h at 37 °C.

All the chemicals were purchased from Sigma-Aldrich, UK, unless stated otherwise. The sperm preparation gradients (PureCeption 40% and 80%) and Quinn’s Advantage Sperm Washing Medium (SWM) were purchased from CooperSurgical, Inc., US. The allophycocyanin (APC)-conjugated Mouse Anti-Human CD46 (Clone E4.3) was purchased from BD Biosciences, Berkshire UK. The AlexaFluor 488 PNA and propidium iodide dye were purchased from ThermoFisher, UK. The progesterone (P4) and prostaglandin E1 (PGE1) were dissolved in DMSO. The final concentration of the DMSO in an experiment is maximally 0.02% v/v. The concentration of the compounds was 10 μM P4, 5 mM PGE1, 30 mM ammonium chloride (NH₄Cl), 10 mM 17α-hydroxyprogesterone (P4OH) and 5 mM prostaglandin E2 (PGE2). The concentrations of the CatSper agonist are at EC₁₀₀ values.

In the co-culture experiments, the iPS-MSCs-CD cells were labeled with copGFP
while HCC1806 cells were labeled with CellTracker Red (catalog number
C34552, Thermo Fisher Scientific, Waltham, MA, USA) as recommended by the
manufacturer. Very briefly, cells were washed with PBS and
10 μM dye was added to one million cells for
30 min. Afterwards, the unattached dye was removed by PBS washing and
centrifugation. For the detection of dead cells, propidium iodide dye
(catalog number P3566, Thermo Fisher Scientific) or trypan blue dye (catalog
number 15250061, Thermo Fisher Scientific) was used according to the
manufacturer’s instructions.

Monocyte-derived DCs (mo-DCs) were generated from purified CD14⁺ monocytes in the presence of IL-4 (25 ng/mL; R&D Systems) and GM-CSF (20 ng/mL; sagramostim [LEUKINE], Genzyme) and matured with LPS (25 ng/mL; MilliporeSigma) as previously described (26 (link)). For antigen-specific T cell stimulation, mature mo-DCs from HLA-A2⁺ donors were pulsed with 1 μg/mL HLA A2–restricted influenza matrix peptide (Flu-MP) (sequence GILGFVFTL, AnaSpec, catalog AS-28310) and cocultured with autologous T cells at a DC/T cell ratio of 1:20 in the presence of IL-2 (20 U/mL). After 1 week, T cells were restimulated with additional Flu-MP–loaded DCs in the presence of IL-2 (20 U/mL), IL-7 (5 U/mL), and IL-15 (5 U/mL). Flu-MP–specific T cells were identified by MHC tetramers (iTag Tetramer HLA-A2 Influenza-M1 [GILGFVFTL], MLB International) and sorted after 2 weeks of culturing (26 (link)). Fresh propidium iodide (PI) dye (10 μg) (Invitrogen, Thermo Fisher Scientific) was used in some experiments to monitor tumor cell lysis. For experiments relating to entry of antigen-specific T cells, antigen-loaded (or unpulsed) DCs were added to tumor colonies 4 hours prior to the addition of T cells. For some experiments, DCs were preincubated with 50 μg/mL α–MHC-1 (clone W6/32, BioLegend) or an isotype control for 1 hour at 37°C prior to peptide loading.