2 nbdg

The fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (Invitrogen) was used as an agent to measure NK cell glucose uptake. Freshly isolated cells were resuspended with prewarmed (37 °C) RPMI-1640 medium (Life Technologies) in the presence of 100 μM 2-NBDG, and cultured at 37 °C for 10 min. Cells were then stained for surface markers (CD3, CD19, NKp46, NK1.1, CD11b, and CD27) before detection via flow cytometry.

The fluorescent glucose analog, 2-NBDG (Invitrogen), was used to measure NK cell glucose uptake. Isolated cells were resuspended in glucose-free medium (Life Technologies) that had been prewarmed (37°C), 3.4 µL of the dissolved 2-NBDG solution added, incubated at 37°C for 30 min and washed with PBS (300 g, 10 min). Before detection by flow cytometry, cells were labeled with surface markers (CD3, CD14, CD19, CD16, CD56, and CD160).

For 2-NBDG staining and sorting, differentiated DE brown adipocytes carrying the Brie CRISPR library were stained with 2-NBDG (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s directions. Briefly, on the morning of staining the induction media were removed and replaced with fresh DMEM. Cells were treated with 100 nM insulin for 15 min, before the addition of 100 uM 2-NBDG. After 1 h incubation at 37 °C in a 5% CO₂ incubator, cells were detached from the plate with Trypsin/EDTA and sorted on a FACSARIA 2 FACS machine (BD Biosciences, San Jose, CA, USA). Just before sorting, propidium iodide was added to the cells at a final concentration of 1 ng/mL (Thermo Fisher, Waltham, MA, USA). All plots were made using FlowJo software (Version 10.8, FlowJo LLC). We sorted approximately 5 × 10⁶ cells from each of the 8 plates to achieve the recommended representation of ~400 cells per sgRNA (4 × 10⁷ total cells sorted). After sorting, gDNA was extracted as previously described. Illumina sequencing was performed by a commercial vendor (Novogene, Nanjing, China). For sorted cells, we acquired a total of 2.98 × 10⁸-reads from 4 × 10⁷ total cells, for approximately 7× read coverage. For preadipocytes and adipocytes, we acquired a total of 1.08 × 10⁸-reads from 3 × 10⁷ total cells, for approximately 3× read coverage.

BP were isolated from E7, E9, E14, and E16 chick embryos in chilled L-15 mediumand subsequently incubated in 1 mM solution of 2-NBDG (N13195, Thermo Fisher Scientific) in L-15 medium at room temperature for 1 hr. The medium was then replaced with a fresh solution of 1 mM 2-NBDG and 350 nm TMRM (T668, Thermo Fisher Scientific) in L-15 and incubated for a further hour at room temperature. Thereafter, the BPs were washed several times with fresh medium containing 350 nM TMRM and mounted in a 3.5-mm glass bottom MatTek dish. 3D image stacks with an optical thickness of 1 μm were captured using a Leica SP5 confocal microscope with an HCX PL APO ×63/1.3 GLYC CORR CS (21°C) objective.