cd8 dendritic cell dc isolation kit  (Miltenyi Biotec)

Journal: Nature

Article Title: SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity.

doi: 10.1038/s41586-023-06299-8

Figure Lengend Snippet: Fig. 1 | Intratumoral glutamine supplementation promotes cDC1-mediated anti-tumour immunity. a, Levels of glutamine and glucose in plasma and TIF of mice bearing MC38 tumours at day 15 (n = 4 per group). b,c, Growth and endpoint weight of MC38 (b; n = 8 per group) and B16-OVA (c; n = 10 per group) tumours (day 24 and 18, respectively) after intratumoral PBS or glutamine supplementation. d, MC38 tumour growth in Rag1−/− mice after PBS or glutamine treatment (n = 7 per group). e, MC38 tumour growth and mouse survival after indicated treatments (n = 12 for Gln + anti-PD-1, n = 13 for all other groups). f, Growth of B16-OVA tumours in mice receiving intratumoral PBS or glutamine with activated OT-I cells (indicated by arrow) (n = 10 per group). g, MC38 tumour growth in tumour-free (having received prior glutamine + anti-PD-1 treatment; n = 8) or naive mice (n = 5) upon challenge with MC38 cells. h, Indicated T cell populations at day 15 in MC38 tumours treated with PBS (n = 7) or glutamine (n = 5). i, DCs, CD45+ non-macrophage immune cells, macrophages and CD45− cells were sorted from PBS- and glutamine-treated MC38 tumours and mixed for scRNA-seq analysis. Violin plots show activity scores of early activation and effector/cytokine signalling signatures in intratumoral CD8+

Article Snippet: Spleens were collected 10 days after tumour inoculation, and cDC1s were enriched using the CD8+ DC isolation kit (130-091-169, Miltenyi Biotec).

Techniques: Clinical Proteomics, Activity Assay, Activation Assay

Journal: Nature

Article Title: SLC38A2 and glutamine signalling in cDC1s dictate anti-tumour immunity.

doi: 10.1038/s41586-023-06299-8

Figure Lengend Snippet: Fig. 2 | Glutamine interplay between tumour cells and cDC1s modulates anti- tumour immunity. a–d, [3H]Thymidine (TdR) incorporation by OT-I (a,c,d) or OT-II (b) cells after coculture with cDC1s (n = 4 per group) pulsed with OVA in amino acid (AA)-replete medium (+AA) or medium lacking an individual amino acid (a,b), in culture supernatants derived from MC38 cells cultured in glutamine- free medium supplemented with indicated glutamine concentrations (c), or in MC38 culture supernatant supplemented with an individual amino acid (d). Arrows in a,b indicate fold change for +AA versus −Gln. e, CD86 and MHCII expression on BMDCs after 24 h of Transwell coculture with MC38 cells in medium containing 2 or 0.6 mM glutamine (n = 3 per group). MFI, mean fluorescence intensity. f, Expression of glutamine transporters in indicated mouse cell types (from GSE121861). NK cells, natural killer cells. g, Immunoblot analysis of SLC38A2 and β-actin in control and SLC38A2-deficient MC38 cells. h, Growth of control and SLC38A2-deficient MC38 tumours in wild-type mice (n = 10 per group). i,j, Indicated T cell populations (i) or IFNγ+, TNF+ or granzyme B+ (GZMB+) CD8+ T cells (j) from control and SLC38A2-deficient MC38 tumours at day 15

Article Snippet: Spleens were collected 10 days after tumour inoculation, and cDC1s were enriched using the CD8+ DC isolation kit (130-091-169, Miltenyi Biotec).

Techniques: Derivative Assay, Cell Culture, Expressing, Fluorescence, Western Blot, Control

Journal: Innate Immunity

Article Title: The second and third amino acids of Pam2 lipopeptides are key for the proliferation of cytotoxic T cells

doi: 10.1177/1753425918777598

Figure Lengend Snippet: The Aa following Pam2Cys determines the cross-priming activity in bone marrow–derived DC (BMDC). (a) and (b) BMDCs were stimulated with Pam2LPs in the presence of OVA. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled OT-I cells were co-cultured with the BMDCs. After 60 h, the degree of OT-I proliferation was assessed by flow cytometry. (a) OT-I cells were gated as CD8α + TCR Vβ5.1,5.2 + population. The histograms of representative samples are shown. (b) The result including all samples is shown. The results are representative of two independent experiments.

Article Snippet: CD8α + DCs (Thy1.2 − B220 − DX5 − CD8α + population) were isolated from mouse spleen with a CD8α + DC isolation kit (Miltenyi Biotec; cat. no. 130-091-169).

Techniques: Activity Assay, Derivative Assay, Cell Culture, Flow Cytometry

Journal: Innate Immunity

Article Title: The second and third amino acids of Pam2 lipopeptides are key for the proliferation of cytotoxic T cells

doi: 10.1177/1753425918777598

Figure Lengend Snippet: Pam2-13s is not directly recognized by OT-I cells. (a) Tlr2 gene expression level on OT-I cells and CD8α + DCs isolated from OT-I mouse were analysed by real-time PCR. Error bars show ±SD. (b) Splenocytes were harvested from OT-I mouse and TLR2. TLR6 expression levels on OT-I cells (CD8α + CD3 + population) and CD8α + DCs (CD8α + CD11c hi population) were analysed by flow cytometry. Shaded and open histograms indicate isotype control and TLR2, TLR6 staining, respectively. (c) CFSE-labelled OT-I cells were cultured singularly in the presence or absence of anti-CD28 and anti-CD3 Abs. PBS, Pam2-13 and Pam2-13s were added to the culture media. After 60 h, the degree of OT-I proliferation was assessed. The histograms of each sample are shown.

Article Snippet: CD8α + DCs (Thy1.2 − B220 − DX5 − CD8α + population) were isolated from mouse spleen with a CD8α + DC isolation kit (Miltenyi Biotec; cat. no. 130-091-169).

Techniques: Gene Expression, Isolation, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Control, Staining, Cell Culture

Journal: Innate Immunity

Article Title: The second and third amino acids of Pam2 lipopeptides are key for the proliferation of cytotoxic T cells

doi: 10.1177/1753425918777598

Figure Lengend Snippet: The conversion of an Aa following Pam2Cys to Serine gives rise to cross-priming in CD8α + DC. (a) and (b) CD8α + DCs (Thy1.2 – B220 – DX5 – CD8α + population) isolated from spleen were stimulated with Pam2LPs in the presence or absence of OVA. CFSE-labelled OT-I cells were co-cultured with the CD8α + DCs. After 60 h, the degree of OT-I proliferation was assessed. (a) The histograms of each sample are shown. (b) The concentration of INF-γ in the culture medium was measured by ELISA. The results are representative of two independent experiments.

Article Snippet: CD8α + DCs (Thy1.2 − B220 − DX5 − CD8α + population) were isolated from mouse spleen with a CD8α + DC isolation kit (Miltenyi Biotec; cat. no. 130-091-169).

Techniques: Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay