Facsaria 3 cell sorter

The following anti-mouse antibodies were used for detection and enrichment of immune cells: APC anti-CD3 (17A2), PE anti-TCRβ (H57–597), APC-Cy7 anti-CD4 (GK1.5), Alexa Fluor^® 488 anti-Foxp3 (R16–715), and PE-Cy7 anti-CD8a (53–6.7) (BD Pharmingen); PE anti-CTLA-4 (UC10–4B9) and PE anti-ICOS (7E.17G9) (eBioscience). PE anti-Ki67 (Biolegend), Anti-EGFR antibody (ab30) (Abcam). For cell surface staining, cells were incubated with corresponding antibodies in PBS for 15 min on ice before analysis on a FACSAria™ III cell sorter (FACSDiva software, BD). Dead cells that stained by propidium iodide (2 μg/ml) were excluded. For intracellular cytokine staining, cells were fixed and permeabilized with BD Cytofix/perm and Perm/wash buffer according to the manufacturer's protocol. Then cells were stained at room temperature for 30 min with Alexa Fluor^® 488 anti-IFN-γ (XMG1.2, Biolegend), Alexa Fluor^® 647 anti-TNF-α (MP6-XT22, Biolegend), APC anti-perforin (eBioOMAK-D, eBioscience) and PE anti-granzyme B (NGZB, eBioscience) respectively before analysis on a BD LSRII flow cytometer. For Foxp3 staining, Foxp3 fix/perm buffer (Biolegend) set was used according to the manufacturer's instruction. All flow cytometry data was analyzed with Flowjo 7.6.1 software. Cell sorting was performed on a FACSAria™ III cell sorter (FACSDiva software, BD) based on cell surface marker staining.

The plasmid encoding Cas9D10A, GFP and gRNAs, the donor plasmid used for recombination, as well as the protocol for validation of the obtained cell line were generated previously (Burov et al., 2021 (link)). SW480 cells were co-transfected with the plasmids using Mirus TransIT-LT1 reagent (Mirus Bio LLC, Madison, WI, USA) according to manufacturer’s instructions. Forty-eight hours post transfection cells were washed with PBS, detached from the plates using trypsin-EDTA solution (Pan-Eko, Moscow, Russia), centrifuged and resuspended in 1 mL of PBS. Then the FACSAria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) was used to obtain the population of cells with considerable GFP fluorescence. After that, cells were cultured for 2 weeks and then stimulated with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF-α (both from R&D systems, Minneapolis, MN, USA) for 72 h. Finally, cells with mCherry fluorescence were collected using the FACSAria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) and propagated as described above.

CD45⁺CD235⁺CD71⁺ cells from peripheral blood mononuclear cells and HCC tissue of one HCC patient were sorted using a FACSAria III cell sorter (BD Bioscience, Franklin Lakes, NJ, USA). CD45⁺TER119⁺CD71⁺ cells and CD45^-TER119⁺CD71⁺ cells from bone marrow (BM), spleen, and tumor tissues from the orthotopic HCC mouse model were also sorted using the FACSAria III cell sorter (BD Bioscience). The sorting purity was > 95%. RNA sequencing was performing using the BGIseq 500 platform (BGI, Shenzhen, China). Single-end read runs were used with read lengths up to 50 bp in high output mode and total read count of 20 M. Data were aligned using RSEM v1.2.12 against the mm10 genome and gene-level read counts, and TPM values at the gene level were estimated for ensemble transcriptome assembly. Samples with at least 80% aligned reads were analyzed. DESeq2 was used to estimate significance between any two experimental groups. Overall changes were considered significant if samples had a p value of < 0.05 with an additional threshold on fold change (FC) ([log₂FC] > 1) taken to generate the final gene set.