7 aad staining

After two days of growth, the cells were collected and spun down at 50 g for 5 min at 15 °C, washed with HBSS (HyClone Laboratories Inc, SH3058802, Logan, UT, USA) and sorted. For the single cell sorting, a suspension of 1 × 10⁶ cells per tube were prepared in 1 mL of HBSS as capture medium. GFP-positive populations were sorted from the transfected clones and non-GFP population was sorted for the control cells (transfected with lentiguides to target GFP) by BD FACSAria cell sorter (BD, Franklin Lakes, NJ, USA). The viability of the cells during the sort was assessed by 7-AAD staining (BioLegend, 420403, San Diego, CA, USA). Single cells were sorted into 96-well plates containing grow media with the total of 2–3 plates per target. After sorting, the plates were maintained at 37 °C humidified incubator with 5% CO₂. Two weeks later the sorted clones were transferred into 12-well plates to expand. Each clone was validated by western blotting when antibodies were available or tested for bactericidal activities upon maturation. Selected clones were maintained in freezing media 80% FBS (Thermo Fisher Scientific, 26140, Waltham, MA, USA) and 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich, 472301, Saint Louis, MO, USA) at 1 × 10⁶ cells per tube. Then the cells were slowly frozen using a cryo-freezing container and stored them at –80 °C.

Pamidronate expanded effector Vδ2 (Eff-Vd2) and Reg-Vδ2 cells were purified (purity > 90%) as described above. Reg-Vδ2 cells were pre-labelled with calcein-AM (100 nM, BioLegend, USA) and co-cultured with Eff-Vδ2 for 48 hours, at the indicated ratios respectively. U937 cells were added afterwards and cultured for additional 4 hours, at 1:10 ratio versus Eff-Vδ2 cells. Then T cells were stained with the fluorochrome-labeled anti-CD3, anti-Vδ2, anti-CD25, anti-CD127, anti-CD38, anti-DNAM1, and anti-NKG2D. Detection for the expression of IFN-γ in Vδ2 cells was same as our previous report (32 (link)). U937 cells were detected by 7-AAD staining (BioLegend, USA).

Immunostaining of GBM cells was performed by incubating cells with antibodies against integrin α10 (Alexa Fluor 647 conjugate), α3, α6, and α7 (see Table S2) for 30 min in the dark at 4 °C prior to flow cytometry analysis using a BD Accuri C6 flow cytometer. After a 30-min incubation with a primary antibody, cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS, SH3002802, Hyclone, Logan, UT, USA) containing 1% FBS and 0.1% sodium azide and incubated with a secondary antibody (Table S3) for 20 min in the dark at 4 °C. For co-staining integrin α3 and α7 with integrin α10, cells were then incubated with integrin α10 Alexa Fluor 647 antibody for 30 min in the dark at 4 °C before subsequent analysis using flow cytometry.
GBM cells with high expression of integrin α10β1 (α10^high) or low expression of integrin α10β1 (α10^low) were sorted by fluorescence-activated cell sorting (FACSAria, BD Biosciences, San Jose, CA, USA). Discrimination of live/dead cells was accompanied by 7-AAD staining (BioLegend, San diego, CA, USA). Sorted cells were washed in medium and re-seeded for recovery and expansion, and some were immediately frozen for RNA extraction.