Zombie uv

A translation-blocking antisense morpholino oligonucleotide complementary to CD47 (CGTCACAGGCAGGACCCACTGCCCA) was obtained from GeneTools as previously described (35 (link)). 4′,6-diamidino-2-phenylindole (DAPI) (Cat#D9542), rat serum (Cat#R9759) and rabbit serum (Cat#R9133) were purchased from Sigma-Aldrich. Aqua live/dead was from ThermoFisher Scientific (Cat#L3495), UV Zombie from BioLegend (Cat#423107), and Ammonium-Chloride-Potassium (ACK) lysis buffer was from Lonza (Cat#10-548E).

Relative telomere length of NK and T cell subsets was assessed as previously described [53 (link)]. Briefly, PBMCs were first stained with a biotinylated anti-CD28 (CD28.A) antibody, followed by Streptavidin-conjugated-Cy3, a fixable live – dead cell stain (UV Zombie, BioLegend) and anti-CD56 (HCD56), anti-CD7 (M-T701), anti-CD3 (OKT3), anti-CD4 (A161A1), anti-CD8 (RPA-T8), anti-CD16 (3G8), anti-CD45RA (HI100), anti-CD27 (LG.3A10). Samples were then washed in PBS, fixed with 1 mM BS3 (Thermo Scientific UK) and quenched with 50 mM Tris–HCl in PBS (pH 7 2, 20 min, room temperature). For the hybridization step, cells were resuspended in 70% deionized formamide, 2.85 mM Tris–HCl pH 7.2, 1.4% BSA and 0.2 M NaCl and 0.75 μg/ml of PNA TelC-Cy5 probe (PNA Bio, US) was added. Samples were then heated for 10 min at 82°C before being rapidly cooled down on ice. After 1 hour of incubation at room temperature, samples were washed twice in 70% deionized formamide, 14.25 mM Tris–HCl pH 7.2, 0.14% BSA, 0.2 M NaCl, 0.14% Tween-20 in 2% BSA/PBS twice before acquisition on a LSRFortessa cytometer (BD Biosciences). Quantum Cy5 molecules of Equivalent Soluble Fluorochrome (MESF) beads (Bangs Laboratories, USA) were acquired alongside samples in each experiment to ensure standardization of FACS machine set up. Data were analyzed using FlowJo®_V10.4 software (Tree Star, Ashland, OR).

All fluorescence associated cell sorting was carried out on a FACS Aria II and analysis on a BD LSR II or a BD Fortessa (Becton Dickinson). The following anti-mouse antibodies were used: CD138-Phycoerythrin (PE), -Allophycocyanin (APC), or -BV510 (281–2, Biolegend) and B220-BV421 (RA3-6B2, Biolegend). When staining cultured cells, propidium iodide (Millipore Sigma) or Zombie UV (Biolegend) were used to exclude dead cells from the analysis. To stain for GLUT1, cells were first fixed in 2% paraformaldehyde (Electron Microscopy Services), permeabilized in 0.1% Saponin (84510, Millipore Sigma) and then stained with an unconjugated GLUT1 monoclonal antibody (SPM498, Thermo Fisher Scientific) followed by a Rat anti-mouse IgG2a -Alexa Fluor 647 detection antibody (SB84a, Southern Biotech). Cells stained with the detection antibody alone were used as an isotype control. Data was analyzed using the FlowJo software (Becton Dickinson). To monitor 2NBDG uptake kinetics, 1x10⁶ cells were resuspended in the appropriate buffer and 2NBDG introduced to the suspension just prior to recording the sample. Mean Fluorescence Intensity (MFI) changes over time were monitored using the Kinetics platform on FlowJo.

Single cell suspensions were washed with PBS and stained for viability with ZombieUV (1:2000; Biolegend) or LIVE/DEAD™ Fixable Aqua (1:500; Invitrogen). Cells were then incubated with 5 μg/ml FC block (αCD16/CD32; 2.4G2; Biolegend) in FACS buffer (PBS containing 2% FBS and 2 mM EDTA), before staining for surface markers for 30 min. After surface staining, cells were washed twice with FACS buffer before fixation in 1% PFA for 10 min. For intracellular staining, cells were permeabilized with eBio Fixation/Permeabilisation buffer for 1 h, before staining with relevant intracellular antibodies for 30 min. Samples were analyzed by flow cytometry (LSR Fortessa, BD) and data analyzed using FlowJo v10 software. A list of antibodies used is provided in Table 1.