Parental (obtained from external vendor such as ATCC, maintained at dedicated internal cell line facility) and PD-L1-deficient MC38 and CT26 (ATCC) colon carcinoma cell lines were maintained in RPMI with 10% fetal bovine serum and 2 mM GlutaMAX (Life Technologies) and routinely tested for mycoplasma contamination (Lonza Mycoalert and Stratagene Mycosensor). Plasmids containing gRNAs and CAS9 were co-transfected into MC38 or CT26 parental cell lines with Lipofectamine LTX with PLUS Reagent (Life Technologies) in Opti-MEM I (Life Technologies). After transient transfection, cells were expanded and stimulated for 48 h with 20 ng ml−1 of mouse interferon-γ (R & D Systems) to induce PD-L1 expression before they were single cell sorted on a BD FACS Aria (BD Biosciences). We confirmed PD-L1-deficient clones by flow cytometry and RT-PCR with TaqMan Assays Mm00452054_m1 (PD-L1 01) and Mm03048247_m1 (PD-L1 03; Life Technologies).
Mouse interferon γ
Manufactured by R&D Systems
Sourced in United States
Mouse interferon-γ is a recombinant protein that represents the mature form of the mouse interferon-gamma cytokine. Interferon-gamma is a key pro-inflammatory cytokine that plays a central role in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Mycoalert, by Lonza (1 mentions)
Trypan blue staining, by Fujifilm (1 mentions)
Murine il 4, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
C57bl 6 mice, by R&D Systems (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
M csf, by R&D Systems (1 mentions)
2 protocols using mouse interferon γ
1
Generation of PD-L1 Deficient Colon Cancer Cell Lines
2
Macrophage Polarization Induction Protocol
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Bone marrow cells from 8-week-old female C57BL/6 mice (SLC Japan, Shizuoka, Japan) were induced into macrophages in DMEM supplemented with 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D Systems, Minneapolis, MN, USA). BMMs were incubated with DMEM alone, 50 ng/mL mouse interferon-γ (IFN-γ; R&D Systems) and 10 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich), 20 ng/mL murine IL-4 (PeproTech, Cranbury, NJ, USA), or SHED-CM for 24 hours designated as M0, M1, M2 (IL-4), and M2 (SHED-CM), respectively. For cell mix experiments, M1 were mixed with M0 at a ratio of 1:1 and subsequently treated with SHED-CM for 24 hours (Fig. 2C ). Their polarization states were examined by flow cytometric analysis and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).
Postinduction, the medium was changed to a serum-free DMEM and incubated for next 24 hours, harvested, and centrifuged at 2000 × g for 5 minutes. The CM from M2 (SHED-CM) or M0 was designated as M2-CM or M0-CM. The protein concentration of each CM was adjusted to 3 µg/mL postharvesting the CM. Cell viabilities of the macrophages were evaluated with trypan blue staining (Fujifilm Wako).
Postinduction, the medium was changed to a serum-free DMEM and incubated for next 24 hours, harvested, and centrifuged at 2000 × g for 5 minutes. The CM from M2 (SHED-CM) or M0 was designated as M2-CM or M0-CM. The protein concentration of each CM was adjusted to 3 µg/mL postharvesting the CM. Cell viabilities of the macrophages were evaluated with trypan blue staining (Fujifilm Wako).
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