Cfda se

We developed a method to label myxobacterial OM proteins with Cy3 (Lumiprobe) to test for transfer between cells. Live cells were grown to mid-log phase, washed, and incubated with Cy3 dyes in appropriate liquid medium as follows: for M. xanthus, 50 µg/ml Cy3 and 1-h incubation in CTT; for S. cellulosum, 1 µg/ml Cy3 and 15-min incubation in medium M. After incubation, the stained cells were washed three to five times in fresh medium and resuspended to ∼5 × 10⁸ cells/ml. Recipient cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Invitrogen) as described previously (5 (link)). Cy3-labeled donors and CFDA-SE-labeled recipients were then mixed (1:1 ratio) and incubated on agarose pads consisting of 1.2% agarose in the appropriate medium. Cell mixtures were incubated in the dark at 33°C for 0.5 h for M. xanthus cells and for ≥4 h for S. cellulosum cells before imaging was performed. To test for OME in C. crocatus Cm c5, a lipid dye transfer assay was done as described previously (9 (link)). Donor cells were labeled with a red fluorescent DiD lipid dye (lipophilic tracer sampler kit; Invitrogen), whereas the recipients were labeled with CFDA-SE (Invitrogen).

Cell permeant dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (65-0850-84, eBioscience, USA) was used to trace proliferation of cells. Staining was done in accordance with the manufacturer's instructions. Briefly, PBMC (1 × 10⁶ cells/ml) were incubated 15 min with 10 µM of CFDA SE (65-0850-84, eBioscience, USA) and washed with RPMI supplemented with 10% FBS, 2 mM l-glutamine.

Cell permeant dye CFDA SE (65-0850-84, eBioscience, San Diego, CA, USA) was used to analyze the leukocyte proliferation according to the manufacturer’s recommendation. Briefly, PBMCs (1 × 10⁶ cells/mL) were incubated with CFDA SE (10 µM; 65-0850-84, eBioscience, San Diego, CA, USA) for 15 min and washed with RPMI supplemented with 10% FBS and 2 mM L-glutamine.

SUM190 cells were labeled at 37 °C with the cell-permeable green fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Thermo Fisher Scientific) at 3.5μM (d0). After washing, CFDA-SE^pos/DRAQ7^neg viable cells were seeded (60,000 cells/well) into 48 well tissue culture plates in full medium (which is required for SUM190 cells to adhere), using a flow cytometer (FACSAria III, BD Biosciences, Scoresby, VIC, Australia) and incubated overnight. The following day the number of viable SUM190 cells (CFDA-SE^pos/DRAQ7^neg) was enumerated by flow cytometry in quadruplicate (FACS Canto II, BD Biosciences) to establish a baseline at d1. The remaining wells containing adherent SUM190 cells were then incubated with either low serum (0.25% FBS) alone, unlabeled viable SUM149 cells (90,000/well) in 0.25% FBS, or unlabeled viable SUM149 cells (90,000/well) in 0.25% FBS plus either the anti-IL-6R antibody Tocilizumab (Roche) or a control humanized IgG1. On d3 the total number of viable SUM190 cells (CFDA-SE^pos/DRAQ7^neg) was again enumerated by flow cytometry (FACS Canto II, BD Biosciences).

For some experiments, cells were restimulated on day 9 after primary stimulation without separating edited and transduced cells. For proliferation assays, the cell mixtures were stained with 2.5 μM carboxyfluorescein diacetate succinimidyl ester (CFDA SE, ThermoFisher, referred to as CFSE) before restimulation with anti-CD3/CD28 beads. For other experiments, cells were separated by FACS on day 9 to purify CD3⁺ and CD3⁻ T cells with or without CAR. For assessing CD25⁺CD71⁺ upregulation, purified CAR T cells were stimulated with parental CD19⁺ Raji cells or CD19^deficient Raji cells for 2 days. In some cultures, CTLA-4 Ig (provided by Dr. Vincenti, UCSF) was added at a concentration of 13.5 μg/ml. For measurements of proliferation, purified cells were stimulated with soluble anti-CD28 (clone CD28.2, 1 μg/ml, BD Pharmigen), plate-bound anti-CD28 (clone CD28.2, 10 μg/ml), or soluble anti-CD3 (clone HIT3α 2 μg/mL. BD Pharmigen). After 48 h, a portion of the supernatant was collected and analyzed for cytokine secretion using multiplexed Luminex (Eve Technologies, Calgary, Canada). The cells were then pulsed with 0.5 μCi of ³H thymidine and cultured for another 16–18 h before harvesting to determine the level of ³H thymidine incorporation using a scintillation counter.

Apoptotic thymocytes were stained with 2.5 µM DeepRed dye (Invitrogen, Carlsbad, CA, USA) for 24 h according to the protocol provided by the manufacturer, while BMDMs were stained with 10μM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Thermo Fisher Scientific, Waltham, MA, USA). Apoptotic thymocytes were added to 2 × 10⁵ C57BL/6 macrophages in 1:5 macrophage:target cell ratio for 1 h, then the remaining cells were washed away. BMDMs were then fixed with 1% paraformaldehyde. Pictures were then taken on a fluorescent microscope (FLoid™ Cell Imaging Station, ThermoFisher, Waltham, MA, USA.