Cell id cisplatin 194pt

Eight fresh early stage NSCLC samples were obtained from the Zhujiang Hospital, Southern Medical University. The detailed clinical characteristics of the samples were shown in Supplementary Table 1. After washing by the RPMI 1640 medium, the fresh lung tumor samples were then dissociated into single cells under Deoxyribonuclease and Collagenase type IV exposure. ACK Lysing Buffer (PLT) was used to remove the erythrocyte, the amounts of viable and dead cells were then counted to provide a preliminary estimate of the sampling efficiency. Cell-ID™ Cisplatin-194Pt (Fluidigm) was used to specifically identify dead cells; next, qualified samples were blocked on ice for 20 minutes. Without removing the blocking solution, samples were incubated with a surface antibody mix (Maxpar^® Antibody Labeling Kit, Fluidigm) for 30 minutes on ice. With Maxpar^® Fix and Perm Buffer, an eventual 500μM DNA intercalator (Cell-ID™ Intercalator-Ir, Fluidigm) were incubated with the washed 200uL re-suspended cells per sample overnight at 4°C. Subsequently, intracellular staining was performed. After washing, pre-fixing, and 30 minutes of co-incubation with intracellular antibody mix on ice, cells were then rinsed and subsequently obtained in the CyTOF system (Helios, Fluidigm) to detect the signals. For the detailed procedures, please refer to the reported protocol (34 (link)).

Isolation of FAPs by MACS technology from mdx mice was performed according to the protocol described in Cerquone Perpetuini et al., 2020.
FAPs were incubated with Cell-ID-Cisplatin-194Pt (Fluidigm, South San Francisco, California, United States, 201194) at the concentration of 1 μM to stain dead cells. Next, samples from three independent biological replicates were barcoded with a combination of palladium isotopes of the Cell-ID 20-Plex Pd Barcoding kit (Fluidigm 201060) at the concentration of 5 μM. Staining was quenched by adding Maxpar cell staining buffer (Fluidigm 201068). Barcoded samples were mixed and stained with lanthanides-conjugated antibodies according to the manufacturer’s instructions. The full list of antibodies purchased from Fluidigm is reported in Supplementary Table 2. Next, the suspension was incubated with Cell-ID Intercalator-Ir (Fluidigm, 201192B) at the concentration of 125 nM for 1 h in Maxpar fix and Perm Buffer (Fluidigm 201067). Finally, cells were filtered with 30 μm cell strainer.
Samples were acquired using CyTOF2 tuned and calibrated according to the manufacturer’s instructions. Rate of acquisition was < 400 events per second. Dataset was converted in fcs files using Debarcoding software (Fluidigm) and normalised.

After washing with RPMI 1640 medium, the fresh lung tumor samples were dissociated into single cells under the irradiation of deoxyribonuclease and type IV collagenase. ACK lysis buffer (PLT) was used to remove the red blood cells, and the number of live and dead cells was then counted to estimate the sampling efficiency. Cell-ID cisplatin 194Pt (Fluidigm) was used to identify the dead cells, after which block qualified samples were placed on ice for 20 min. Each sample was then incubated on ice for 30 min, with the surface antibody mixture (Maxpar Antibody Labeling Kit; Fluidigm) and without removing the blocking solution, using Maxpar Fix and Perm Buffer. The final 500 μMNA intercalator (Cell-ID Intercalator-Ir; Fluidigm) was incubated with 200 μl after the resuspended cells were washed in each sample and finally stored overnight at 4°C. Subsequently, intracellular staining was performed, the cells were washed with the intracellular antibody mixture on ice, pre-fixed, and co-incubated for 30 min. Then the cells were rinsed and then collected on the CyTOF system (Helios; Fluidigm) to detect the signal (Han et al., 2018 (link)). Antibody selection is shown in Supplementary Table 1.