Celltrace violet ctv proliferation kit

Flow cytometric analysis was performed using Beckman Coulter Gallios Flow Cytometer (Beckman Coulter, CA, USA). CMs were pre-stained using Cell Trace Violet (CTV) proliferation kit (Invitrogen, CA, USA) according to the manufacturer's protocol and were treated with their respective doses of NAC and DOX, as described earlier. On day 1 and day 3, the 3D spheroidal droplets with cells were cut using a blade, and cells were extracted using Miltenyi gentleMACS Dissociator (Miltenyi Biotec, MA, USA) using a Multi tissue Dissociation Kit-1 by running the Multi_B program according to the manufacturer's protocol. For 2D samples, cells were detached using trypsin-EDTA. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, then added to their assigned FACS analysis falcon tubes, and analysed using excitation and emission wavelengths of 405 and 450 nm, respectively. Negative controls included freshly isolated non-stained cells and positive controls were pre-stained with CTV.^{1,5,32 (link)}

To track proliferation in response to antigen stimulation, PBMCs were labeled using the Cell Trace^® Violet (CTV) proliferation kit (Invitrogen, Eugene, OR) as described in (1 (link)) with minor modifications. Briefly, CTV dye was reconstituted as recommend by the manufacturer with 20μl of provided DMSO, resuspended in 780 μl of DPBS and then diluted 1:10 in DPBS. A suspension of 1.5 × 107 cells in 1 ml of DPBS were then treated with 150 μl of the 1:10 CTV dye stock, for a final CTV dilution of 1:66. Cells were incubated at room temperature for 20 min. Cells were then washed, centrifuged, and resuspended in 1.5mL of cRPMI media.

Furfuryl gelatin (f-gelatin), used as a basis for the “bioink”, was synthesized and characterized as described [15 (link),16 (link)]. Hyaluronic Acid Sodium Salt (HA; mol. wt. ~1.5–1.8 × 10⁶ Da) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Rose Bengal (RB), a visible light crosslinker, was procured from ThermoFisher Scientific (Waltham, MA, USA). Pluronic F127 was obtained as a “bioink” from Allevi (formerly Biobots, Philadelphia, PA, USA). 1X PBS was obtained as a sterile solution (ThermoFisher, Waltham, MA, USA). The Live/Dead Cytotoxicity Assay Kit Green/Red Staining kit was obtained and used following the manufacturer’s recommendations (Marker Gene Technologies Inc., Eugene, OR, USA). Dulbecco Modified Eagle’s Medium (DMEM) and 0.25% trypsin-EDTA were obtained from ThermoFisher. The Cell Trace Violet (CTV) proliferation kit was obtained from Invitrogen (Carlsbad, CA, USA). Cells and growth medium used in this study are enlisted in the subsequent section.

After thawing, CD34⁺ cells of three independent healthy donors were labeled separately using CellTrace Violet (CTV) Proliferation Kit (Invitrogen) to allow tracking of multiple generations with dye dilution. A sample of those parental generation cells were used to measure fluorescence intensity at time point 00 h. The other cells were incubated according to the conditions described earlier. At 96 h, cells were harvested and labeled using CD133-APC (clone AC133, dilution 1:50; Miltenyi Biotech) and CD34-PE (clone AC136, dilution 1:10; Miltenyi Biotech) cell surface antibodies after a non-specific sites’ saturation step with Gamma Immune (dilution 1:2; Sigma-Aldrich). Isotype controls (Miltenyi Biotech), VersaComp compensation beads (Beckman Coulter), and negative controls were used for subsequent gating strategy. Viability was controlled using a 7-aminoactinomycin D marker (Invitrogen). The fluorescence intensity of CTV and antibodies was measured with the SP6800 cytometer (Sony) from the ImCy platform (Généthon).