- PMID: 35641483
To better understand the TIME of BP tumors, we performed tissue-based multiplexed cyclic immunofluorescence (CyCIF) analysis.
免疫細(xì)胞流式標(biāo)記物
1）We found that BP tumors are highly infiltrated by CD45+ immune cells, of which myeloid cells (CD11b+, F4/80+ or CD11c+), such as F4/80+ macrophages, are more abundant than T lymphocytes (CD4+ or CD8+)
2）tumor-associated macrophages (TAMs; CD45+ CD11b+ F4/80+)，M2-like (MHC-IILow CD206+)
3）CD163 (an M2 marker) and reduction of CD86 (an M1 marker)
4）genes associated with pro-tumorigenic M2 polarization, including Arg1, Csf1r, Il1b and Mrc1, but also strongly stimulated the expression of genes associated with an anti-tumorigenic M1 signature (e.g., Ccl5, Cxcl10, Cd40, Cd86, Il18 and Nos2)
Mouse macrophages were derived from the bone marrow (BM) of FVB/N mice by modifying previously described protocols（PMID: 21356739）.
小鼠缺乏Macrophage colony-stimulating factor (M-CSF) ，培養(yǎng)需要額外加入倍试，In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium.也可以購(gòu)買10?ng/mL mouse M-CSF (BioLegend, # 576404).
THP-1 macrophages 人來(lái)源的巨噬細(xì)胞系
條件培養(yǎng)基收集的步驟
For preparation of tumor cell-conditioned media (CM), tumor cells were grown to 60% of confluence, washed twice with PBS, and then incubated with fresh DMEM with or without drugs for two days. CM were then harvested and centrifuged to collect the supernatant.
從組織中獲取單細(xì)胞懸液
To obtain single-cell suspensions, tumors were excised, minced and dissociated in collagenase/hyaluronidase buffer [DMEM with 5% FBS, 10?mM HEPES (Gibco), 100?μg/mL penicillin–streptomycin, 20?μg/mL DNase I (StemCell) and 1× collagenase/hyaluronidase (StemCell)] for 45?min at 37 °C with agitation, followed by treatment with ammonium-chloride-potassium (ACK) buffer (Lonza) for red blood cell (RBC) lysis, and strained through a 70?μm strainer to remove undigested tumor tissues.
Spleens and tumor-draining lymph nodes (TDLNs) were mechanically dissociated by passing the tissues through a 70?μm strainer using the plunger of a 5?mL syringe（注射器）, and RBCs were lysed as described above.
跑流式組織的暫時(shí)保存
Cells were stained in cold FACS buffer (PBS containing 0.2% BSA and 5?mM EDTA) with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher) for 30?min on ice.
Quantification of cytosolic DNA
Approximately 1×107 cells were lysed and the nuclear, cytosolic, and mitochondrial fractions were obtained using the mitochondrial isolation kit (Thermo Fisher – 89874). Protease inhibitors were not used to enable subsequent DNA purification. Mitochondria were purified at 12,000xg to minimize their contamination in the cytosolic fraction. DNA was subsequently isolated from the nuclear, cytosolic, mitochondrial fractions using the Qiagen DNeasy blood and tissue kit (Qiagen – 69506) and dsDNA was quantified using Qubit 2.0 (Invitrogen) using Qubit dsDNA HS Reagent.
DMXAA, a potent murine STING agonist
