Journal: bioRxiv

Article Title: Tumor-Intrinsic Response to IFNγ Shapes the Tumor Microenvironment and Anti-PD-1 Response in NSCLC

doi: 10.1101/531236

Figure Lengend Snippet: LLC-NT or LLC-sh21 ( Socs1 knockdown) cells were injected into the left lung lobes of mice. After 2 weeks, mice were sacrificed and their tumor-bearing lung lobes were isolated for either flow cytometry or FFPE and T cell staining by immunofluorescence. Representative images of T cell staining from (A) A LLC-NT tumor or (B) A LLC-sh21 tumor at 40X magnification (Scale Bar 100µm) showing CD3 + cells (green), CD8 + cells, (red) and DAPI (blue). Quantification of (C) CD3 + T cells and (D) CD8 + T cells per high power field (HPF) in LLC-NT versus LLC-sh21 tumors (6 tumors in each group). T cell numbers per HPF were averaged over 4 experiments (6 random fields per tumor × 4 staining experiments=24 fields averaged/tumor in total). (E-H) Flow cytometry analysis of T cells was performed using 3 experimental replicates × 3 tumor bearing lung lobes each, for a total of 9 lung lobes per the experimental conditions of “LLC-NT” or “LLC-sh21”. (E) the percentage of PD-1 expressing CD8 + T cells, (F) double-positive PD-1/CD69 expressing CD8 + T cells gated as a percentage of all CD8 + T cells. (G-H) Single cell suspensions were either unstimulated (treated with Brefeldin and Monensin alone) or stimulated (treated with Brefeldin, Monensin, and PMA/Ionomycin) for 5 hours and analyzed by flow cytometry for (G) the percentage of single positive IFNγ expressing CD8 + T cells or (H) double-positive IFNγ/TNFα expressing CD8 + T cells gated as a percentage of all CD8 + T cells. Empty circles represent the LLC-NT condition while the solid black squares represent the LLC-sh21 condition. Error bars represent the mean of the data ±SEM after a student’s t-test (6C-F) or a two-way ANOVA (6G-H) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Antibodies and Reagents: 89Y-CD45, Fluidigm, Clone 30-F11; 141Pr-Gr1 (Ly6C/Ly6G), Fluidigm, Clone RB6-8C5; 142Nd-CD11c, Fluidigm, Clone N418; 143Nd-GITR, Fluidigm, Clone DTA1; 144Nd-MHC class I, Fluidigm, Clone 28-14-8; 145Nd-SiglecF-PE/anti-PE, BD, Clone E50-2440/ Fluidigm, Clone PE001; 146Nd-CD8a, Fluidigm, Clone 53-6.7; 147Nd-p-H2AX[Ser139], Fluidigm, Clone JBW301; 148Nd-CD11b, Fluidigm, Clone M1/70; 149Sm-CD19, Fluidigm, Clone 6D5; 150Nd-CD25, Fluidigm, Clone3C7; 151Eu-CD64, Fluidigm, Clone X54-5/7.1; 152Sm-CD3e, Fluidigm, Clone 145-2C11; 153Eu-PD-L1, Fluidigm, Clone 10F.9G2; 154Sm-CTLA4, Fluidigm, Clone UC10-4B9; 155Gd-IRF4, Fluidigm, Clone 3E4; 156Gd-CD90.2(Thy-1.2), Fluidigm, Clone 30-H12; 158Gd-FoxP3, Fluidigm, Clone FJK-16s; 159Tb-PD-1, Fluidigm, Clone RMP1-30; 160Gd-CD80/86-FITC/anti-FITC, BD, Clone 16-10A1/BD Clone BL1/Fluidigm, Clone FIT22; 161Dy-INOS, Fluidigm, Clone 4B10; 162Dy-Tim3, Fluidigm, Clone RMT3-23; 163Dy-CXCR3-APC/anti-APCCXCR3-173, Biologend/Fluidigm, Clone APC003; 164Dy-IkBa, Fluidigm, Clone L35A5;165Ho-Beta-catenin (active), Fluidigm, Clone D13A1; 166Er-Arg1, Fluidigm, Clone 6D5; 167Er-NKp46, 2 Fluidigm, Clone 9A1.4; 168Er-Ki-67; Fluidigm, Clone Ki-67; 169Tm-Ly-6A/E (Sca-1), Fluidigm, Clone D7; 170Er-CD103-Biotin/anti-Biotin, Biolegend, Clone 2E7/Fluidigm, Clone 1D4-C5; 171Yb-CD44, Fluidigm, Clone IM7; 172Yb-CD4, Fluidigm, Clone RM4-5; 173Yb-CD117 (ckit), Fluidigm, Clone 2B8; 174Yb-Lag3, Fluidigm, Clone M5/114.15.2;175Lu-CD127, Fluidigm, Clone A7R34; 176Yb-ICOS, Fluidigm, Clone 7E.17G9; 191Ir, 193Ir-Intercalator, Cell-ID; 195Pt Cisplatin 5 uM, Cell-ID; 140Ce, 151Eu, 153 Eu, 165Ho, 175Lu Normalization Beads; Cell ID 20-Plex Pd Barcoding Kit, Fluidigm,#201060 (102, 104, 105, 106, 108, 110 Pd Bar Codes); Benzonase, Sigma #E1014-5KU(1:10,000) in HBSS.

Techniques: Injection, Isolation, Flow Cytometry, Staining, Immunofluorescence, Expressing

Journal: Immunity

Article Title: Negative costimulation constrains T cell differentiation by imposing boundaries on possible cell states

doi: 10.1016/j.immuni.2019.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-CD8a 146Nd , Fluidigm , Cat #:3146003B; Clone:53-6.7.

Techniques: Virus, Recombinant, Adjuvant, Polymer, Conjugation Assay, Staining, Mass Cytometry, Sequencing, Transgenic Assay, Double Knockout, Software

Journal: Current protocols in molecular biology

Article Title: Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis Current Protocols in Molecular Biology UNIT 25C.1

doi: 10.1002/cpmb.37

Figure Lengend Snippet: Tissue-specific antibody panels

Article Snippet: This protocol was adapted from previously established fluorescence flow cytometry protocols ( Krutzik et al., 2005 ; Krutzik et al., 2004 ; Leelatian et al., 2015 ; McLaughlin et al., 2008 ). table ft1 table-wrap mode="anchored" t5 caption a7 Metal Antibody Clone Working conc (μg/mL) Dilution Volume to use in 100 μL stain (μL) Sample type Staining condition Ton Glio Mel Surf Sap MeOH 141Pr HLA-ABC W3-32 Fluidigm 1:200 0.5 ✓ ✓ 142Nd cCasp3 D3E9 Fluidigm 1:200 0.5 ✓ ✓ ✓ ✓ 144Nd CD11b ICRF44 Fluidigm 1:200 0.5 ✓ ✓ 145Nd CD4 RPA-T4 Fluidigm 1:200 0.5 ✓ ✓ CD31 WM59 Fluidigm 1:200 0.5 ✓ ✓ ✓ 146Nd IgD IA6-2 Fluidigm 1:200 0.5 ✓ ✓ CD64 10.1 Fluidigm 1:200 0.5 ✓ ✓ CD8a RPA-T8 Fluidigm 1:200 0.5 ✓ ✓ 147Sm β-catenin D10A8 Fluidigm 1:200 0.5 ✓ ✓ 148Nd CD16 3G8 Fluidigm 1:200 0.5 ✓ ✓ 149Sm CD45RO UCHL1 Fluidigm 1:200 0.5 ✓ ✓ ✓ ✓ 152Sm TUJ1 TUBB3 50 1:100 1 ✓ ✓ 153Eu CD45RA HI100 Fluidigm 1:200 0.5 ✓ ✓ S100B 19-S100B 100 1:100 1 ✓ ✓ ✓ 154Gd CD45 HI30 Fluidigm 1:400 0.25 ✓ ✓ ✓ ✓ 155Gd CD27 L128 Fluidigm 1:100 1 ✓ ✓ 156Dy Vimentin RV202 Fluidigm 1:200 0.5 ✓ ✓ 159Tb CD11c Bu15 Fluidigm 1:200 0.5 ✓ ✓ CD49F GoH3 100 1:100 1 ✓ ✓ 160Gd CD14 M5E2 Fluidigm 1:200 0.5 ✓ ✓ 161Dy CD19 HIB19 100 1:100 1 ✓ ✓ PDGFRα 16A1 200 1:100 1 ✓ ✓ Cytokeratin C-11 Fluidigm 1:200 0.5 ✓ ✓ 162Dy c-MET L6E7 100 1:100 1 ✓ ✓ 163Dy SOX2 O30-678 100 1:100 1 ✓ ✓ ✓ 166Er CD24 ML5 Fluidigm 1:200 0.5 ✓ ✓ 167Er CD38 HIT2 Fluidigm 1:200 0.5 ✓ ✓ 168Er CD8 SK1 Fluidigm 1:200 0.5 ✓ ✓ Nestin 10C2 100 1:100 1 ✓ ✓ 169Tm CD44 BJ18 100 1:100 1 ✓ ✓ ✓ 170Er CD3 SP Fluidigm 1:200 0.5 ✓ ✓ ✓ ✓ 171Yb CD68 Y1/82A Fluidigm 1:200 0.5 ✓ ✓ GFAP 1B4 25 1:100 1 ✓ ✓ 172Yb IgM MHM-88 Fluidigm 1:200 0.5 ✓ ✓ 173Yb αSMA Ab54723 50 1:100 1 ✓ ✓ ✓ 174Yb HLA-DR L243 Fluidigm 1:200 0.5 ✓ ✓ ✓ ✓ 175Lu CD56 HCD56 50 1:100 1 ✓ ✓ ✓ ✓ 176Yb Histone H3 D1H2 Fluidigm 1:200 0.5 ✓ ✓ ✓ ✓ Open in a separate window Conc = concentration; Ton = tonsil; Glio = glioma; Mel = melanoma; Surf = surface; Sap = saponin; MeOH = post-methanol Fluidigm = use antibodies provided by Fluidigm Tissue-specific antibody panels

Techniques: Staining

Journal: Immunity

Article Title: Pro-inflammatory Aorta-Associated Macrophages Are Involved in Embryonic Development of Hematopoietic Stem Cells

doi: 10.1016/j.immuni.2019.05.003

Figure Lengend Snippet:

Article Snippet: Anti-Mouse CD43 (clone S11)-146Nd , Fluidigm , Cat# 3146009C.

Techniques: Purification, Recombinant, Software, Control, High Throughput Screening Assay, Sequencing

Journal: Frontiers in Oncology

Article Title: Tumor-Infiltrating Cytotoxic T Cells and Tumor-Associated Macrophages Correlate With the Outcomes of Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer

doi: 10.3389/fonc.2021.743540

Figure Lengend Snippet: Antibody panel.

Article Snippet: CD16 , Fluidigm , 3146020D.

Techniques:

Journal: Cell reports

Article Title: Characterization of HIV-induced remodeling reveals differences in infection susceptibility of memory CD4 + T cell subsets in vivo

doi: 10.1016/j.celrep.2021.109038

Figure Lengend Snippet:

Article Snippet: CD8 , RPA-T8 , 146Nd , Fluidigm.

Techniques:

Journal: iScience

Article Title: The immune landscape of SARS-CoV-2-associated Multisystem Inflammatory Syndrome in Children (MIS-C) from acute disease to recovery

doi: 10.1016/j.isci.2021.103215

Figure Lengend Snippet:

Article Snippet: Anti-IgD (clone IA6-2, conjugated to 146Nd) , Fluidigm , Cat#3146005B.

Techniques: Purification, Clinical Proteomics, Recombinant, Binding Assay, Blocking Assay, Staining, Saline, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Software, Cytometry