monomac1 cells  (Corning Life Sciences)

Journal: Nature Medicine

Article Title: A phase 1/2 trial of an immune-modulatory vaccine against IDO/PD-L1 in combination with nivolumab in metastatic melanoma

doi: 10.1038/s41591-021-01544-x

Figure Lengend Snippet: a , Left, PD-L1-specific T cell culture (MM1636.05) reactivity against PD-L1 peptide or autologous tumor cells in the IFN-γ ELISPOT assay. Tumor cells were either not treated or pretreated with 200 U ml −1 IFN-γ for 48 h before the assay. Effector:target (E:T) ratio of 10:1 was used. Right, PD-L1 and HLA-II surface expression on melanoma cells with (green) or without (yellow) pretreatment with IFN-γ compared to an isotype control (gray) as assessed by flow cytometry. b , Left, PD-L1-specific T cell (MM1636.05) reactivity in the IFN-γ ELISPOT assay against autologous tumor cells pretreated with IFN-γ (500 U ml −1 ) and transfected with mock or PD-L1 small interfering (si)RNA 24 h after transfection. E:T ratio, 10:1. Right, PD-L1 surface expression on melanoma tumor cells (MM1636.05) assessed by flow cytometry 24 h after transfection with mock (blue) or PD-L1 (red) siRNA compared to the isotype control (gray). c , Reactivity of the CD4 + PD-L1-specific T cell clone (MM1636.14) against PD-L1 peptide or autologous CD14 + cells; E:T ratio, 10:1. CD14 + cells were isolated using magnetic bead sorting and used as targets in an ELISPOT assay directly or after pretreatment for 2 d with TCM derived from the autologous tumor cell line. d , Quantitative PCR with reverse transcription (RT–qPCR) analysis of PD-L1 ( CD274 ) expression in sorted CD14 + cells before and after treatment with autologous TCM for 48 h. e , Reactivity of the IDO-specific CD4 + T cell clone (MM1636.23) against IDO peptide combined with HLA-DR (L243)-, HLA-DQ (SPV-L3)- or HLA-DP (B7/21)-blocking antibodies (aHLA-DR, aHLA-DQ and aHLA-DP) in an intracellular staining assay (ICS) for IFN-γ and TNF-α production. T cells were incubated with individual blocking antibodies (2 μg ml −1 ) for 30 min before adding IDO peptide. f , Reactivity of the IDO-specific CD4 + T cell clone (MM1636.23) against the HLA-DR-matched IDO-expressing cell line MonoMac1 transfected with mock or IDO siRNA in an ICS assay, E.T ratio, 4:1. siRNA transfection was performed 48 h before the experiment. g , RT–qPCR analysis of IDO1 expression in MonoMac1 cells 48 h after siRNA transfection. h , Reactivity of the CD4 + IDO-specific T cell clone (MM1636.14) against IDO peptide or autologous CD14 + cells; E:T ratio, 20:1. CD14 + cells were isolated using magnetic bead sorting and used as targets in an ELISPOT assay directly or after pretreatment with TCM derived from the autologous tumor cell line. i , RT–qPCR analysis of IDO1 expression in sorted CD14 + cells before and after treatment with autologous TCM for 48 h. Bars in RT–qPCR data ( d , g , i ) represent the mean of three ( d , i ) or six ( g ) technical replicates ±s.d.; P values were determined by two-tailed parametric t -tests. ELISPOT counts ( a , b , c , h ) represent the mean value of three technical replicates ±s.e.m.; response P values were determined using the distribution-free resampling (DFR) method. TNTC, too numerous to count.

Article Snippet: The acute monocytic leukemia cell line MonoMac1 was obtained from the DSMZ—German Collection of Microorganisms and Cell Cultures (ACC 252) and cultured in RPMI 1640 with GlutaMAX, 25 mM HEPES, pH 7.2 (Gibco, 72400-021) and 10% heat-inactivated FBS.

Techniques: Cell Culture, Enzyme-linked Immunospot, Expressing, Control, Flow Cytometry, Transfection, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Reverse Transcription, Quantitative RT-PCR, Blocking Assay, Staining, Incubation, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Extracellular polyphosphate signals through Ras and Akt to prime Dictyostelium discoideum cells for development

doi: 10.1242/jcs.203372

Figure Lengend Snippet: Polyphosphate decreases proteasome activity. (A) Cells were cultured with the indicated concentrations of polyphosphate for 4 h and proteasome activity levels were measured and normalized to no-polyphosphate (polyP) controls. (B,C) Cells were cultured with 150 µM polyphosphate for 4 h and proteasome subunit 5 levels were measured by western blotting (a representative image of four blots is shown) and normalized to no-polyphosphate controls. A longer exposure (middle) showed no additional bands. Total protein loading control from an aliquot of the samples used for the proteasome subunit 5 western blot (representative image of four gels) is also shown (right). (D) Human leukemia cell lines were cultured with 0 (control) or 150 µM polyphosphate for 24 h and proteasome activity levels were measured. Differences between the various control values were not statistically significant (one-way ANOVA, Tukey's test). *P<0.05, ***P<0.001 (t-tests). (E) Cells were cultured with the indicated amount of polyphosphate for 24 h and cell counts were normalized to the no-polyphosphate control, so that 100% proliferation is the amount of proliferation with no polyphosphate, and 0% proliferation represents no increase in cell number. 150 and 300 µM polyphosphate significantly decreased cell numbers compared to no polyphosphate for MM1 (P<0.05 for both 150 and 300 µM), HL-60 (P<0.01 and P<0.001, respectively), K562 (P<0.01 and P<0.001, respectively), THP-1 (P<0.01 and P<0.001, respectively) and U937 (P<0.01 and P<0.001, respectively). *P<0.05, **P<0.01, ***P<0.001 (t-tests). All values are mean±s.e.m., n≥3.

Article Snippet: MonoMac1 (MM1) cells were from DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) ( Ziegler-Heitbrock et al., 1988 ; Tsuchiya et al., 1980 ; Sundström and Nilsson, 1976 ; Klein et al., 1976 ; Gallagher et al., 1979 ).

Techniques: Activity Assay, Cell Culture, Western Blot, Control