Journal: Theranostics

Article Title: Single-cell RNA sequencing highlights the immunosuppression of IDO1 + macrophages in the malignant transformation of oral leukoplakia

doi: 10.7150/thno.99112

Figure Lengend Snippet: Annotation and distribution of macrophage subpopulations in PCA, preca-OLK and OSCC (A) UMAP plot showing the composition of macrophage colored by cluster. (B) Bar plot showing proportion of each macrophage subtype in each tissue. (C) Statistical analysis comparing the proportions of IDO1 + CD68 + cells (IDO1 + macrophages) in tissues of OLK (n = 25), preca-OLK (n = 15) and OSCC (n = 11). (D) Representative IF staining of human tissues (40x). DAPI (blue), IDO1 (red), CD68 (green). Bar, 50 μm. (E) Heatmap showing representative GO pathways enrichment of the gene set expressed in macrophage subsets predicted by GSVA. (F) Violin plot showing the immunosuppression scoring of macrophage subsets. (G) Heatmap showing the expression of immunosuppressive ligand and receptor molecules in macrophage subsets in PCA, preca-OLK and OSCC.

Article Snippet: Briefly, the sections were labeled with primary antibodies anti-IDO1 (Proteintech, 13268-1-AP), anti-CD68 (Abcam, ab955), anti-Pan-CK (ZSGB-BIO, ZM-0069), anti-CD3 (ZSGB-BIO, ZM-0417), anti-CD8 (ZSGB-BIO, ZA-0508), and anti-PD-1 (ZSGB-BIO, ZM-0381), followed by HRP-conjugated secondary antibody.

Techniques: Staining, Expressing

Journal: Theranostics

Article Title: Single-cell RNA sequencing highlights the immunosuppression of IDO1 + macrophages in the malignant transformation of oral leukoplakia

doi: 10.7150/thno.99112

Figure Lengend Snippet: IDO1 + macrophages exert immunosuppressive effects. (A) Heatmap showing the interacting relationship pairs between macrophage subsets and T cell subsets according to CellPhoneDB analysis in preca-OLK. (B) Dot plot showing the interaction intensity of immune checkpoint ligand/receptors between macrophage subsets and CD4/CD8_exhuausted according to CellPhoneDB analysis. (C) mIF results showing the spatial distribution of PD-1 + CD3 + CD8 + cells (exhausted CD8 + T cells) around IDO1 + CD68 + cells (IDO1 + macrophages), red area indicates the area with a radiu≤50 μm, as green area indicates the area with a radiu >50μm from the IDO1 + CD68 + cells (IDO1 + macrophages). (D-E) . Representative image showing the spatial distribution of PD-1 + CD3 + CD8 + cells (exhausted CD8 + T cells) in the area ≤50 μm from the IDO1 + CD68 + cells (IDO1 + macrophages) in preca-OLK (D) and OSCC (E) . White arrows indicate examples of IDO1 + CD68 + cells; yellow arrows indicate examples of PD-1 + CD3 + CD8 + cells.

Article Snippet: Briefly, the sections were labeled with primary antibodies anti-IDO1 (Proteintech, 13268-1-AP), anti-CD68 (Abcam, ab955), anti-Pan-CK (ZSGB-BIO, ZM-0069), anti-CD3 (ZSGB-BIO, ZM-0417), anti-CD8 (ZSGB-BIO, ZA-0508), and anti-PD-1 (ZSGB-BIO, ZM-0381), followed by HRP-conjugated secondary antibody.

Techniques:

Journal: Theranostics

Article Title: Single-cell RNA sequencing highlights the immunosuppression of IDO1 + macrophages in the malignant transformation of oral leukoplakia

doi: 10.7150/thno.99112

Figure Lengend Snippet: IDO1 is upregulated in HNSC, and activation of the IFN-γ-JAK-STAT axis is the potential cause for differentiation to IDO1 + macrophages. (A) Umap plot showing the differentiation trajectory of macrophages and the distribution of each cell subset on the trajectory. (B) Comparison of IDO1 expression between tumor (n = 519) and normal (n = 44) in TCGA database. (C) The boxes showing the expression distribution of IDO1 in PCA (G1), OLK (G2) and OSCC (G3) in GEO data. (D) Gene set enrichment analysis (GSEA) results showing the enrichment of JAK-STAT gene sets in Macro_IDO1. (E) Information flow of feature signaling pathway in different tissues. (F) The CellPhoneDB communication analysis demonstrating that IFN-II signaling pathway mediates communication between T cells and macrophages in preca-OLK and OSCC. (G) Scatter plot showing the correlation between IFNG and IDO1 expression across 4 independent datasets with OLK in GEO data. (H) Heatmap showing RAS activity of regulons in macrophage subtype predicted by SCENIC.

Article Snippet: Briefly, the sections were labeled with primary antibodies anti-IDO1 (Proteintech, 13268-1-AP), anti-CD68 (Abcam, ab955), anti-Pan-CK (ZSGB-BIO, ZM-0069), anti-CD3 (ZSGB-BIO, ZM-0417), anti-CD8 (ZSGB-BIO, ZA-0508), and anti-PD-1 (ZSGB-BIO, ZM-0381), followed by HRP-conjugated secondary antibody.

Techniques: Activation Assay, Comparison, Expressing, Activity Assay

Journal: Theranostics

Article Title: Single-cell RNA sequencing highlights the immunosuppression of IDO1 + macrophages in the malignant transformation of oral leukoplakia

doi: 10.7150/thno.99112

Figure Lengend Snippet: The IDO1 inhibitor significantly reduces 4NQO induced oral carcinogenesis in mice (A) Schematic plot showing the induction of OSCC by 4NQO in C57BL/6 mice. (B) Macroscopic lesions on the tongues of each group of mice. The dotted circles indicate macroscopic cauliflower-like lesions. (C) Statistical results of macroscopic lesions on the tongues of each group of mice. (D) Statistical results of the tongue lesions (carcinoma) in mice of each group. (E) Representative microscopic images of typical pathological images following H&E staining. Scale bar: 100 μm. (F) Graphical illustration of the working model. Abbreviations: OLK, oral leukoplakia; preca-OLK, precancerous OLK; OSCC, oral squamous cell carcinoma; Trp, tryptophan; Kyn, kynurenine.

Article Snippet: Briefly, the sections were labeled with primary antibodies anti-IDO1 (Proteintech, 13268-1-AP), anti-CD68 (Abcam, ab955), anti-Pan-CK (ZSGB-BIO, ZM-0069), anti-CD3 (ZSGB-BIO, ZM-0417), anti-CD8 (ZSGB-BIO, ZA-0508), and anti-PD-1 (ZSGB-BIO, ZM-0381), followed by HRP-conjugated secondary antibody.

Techniques: Staining

Journal: Mucosal Immunology

Article Title: Intestinal CD103 + CD11b − dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells

doi: 10.1038/mi.2015.64

Figure Lengend Snippet: Epithelial expressed interferon-γ (IFN-γ)-inducible genes are strongly affected by ablation of CD103 + CD11b − dendritic cells (DCs). ( a ) Heat map showing differential expression of selected genes regulated by IFN-γ of colon intestinal epithelial cells (IECs) obtained from wild-type (WT) untreated mice and dextran sodium sulfate (DSS)-treated day 4 WT and Clec9A–diphtheria toxin receptor (DTR) mice ( n =3). ( b ) Gene validation comparing bulk IECs and CD45 + lymphocyte-depleted IECs obtained from DSS-treated animals. IECs were isolated from the colon as described in Methods and loaded on a Percoll gradient to separate the lymphocytes from the epithelial fraction. RNA and subsequently complementary DNA (cDNA) was prepared and validated for Cd3 , Ifn- γ, and a series of Ifn- γ - induced genes, including Ido1 and IL-18bp. One representative sample is shown. ( c ) Quantitative real-time PCR (qPCR) analysis of Ido1 expression in different intestinal DC subsets and IECs at steady state (SS) and 4 days after DSS treatment. N =3±s.e.m. ( d ) Indoleamine 2,3 dioxygenase (IDO1) is the major tryptophan-degrading enzyme in the colonocytes. IECs obtained from distal part of the colon of DSS-treated WT mice (day 4) were analyzed for Ido1 , Ido2 , and Tdo expression by semiquantitative real-time PCR (RT-PCR) analysis. Hprt was used as an endogenous mRNA control. Results are representative of 3–4 pooled colons. ( e ) Ido1 and IL-18bp expression profile during DSS treatment in IECs. WT mice were treated with 1% DSS over 6 days. Colonocytes were isolated from the distal part of three mice every day and monitored by RT-PCR for Ido1 and IL-18bp mRNA expression. ( f ) qPCR analysis of IL-18bp expression in IECs at steady state and 4 days after DSS treatment. N =3±s.e.m. ( g ) RT-PCR analysis of Ido1 and IL-18bp in IECs obtained from pooled colons of DT-injected untreated or DSS-treated (day 4) WT, Clec9A-DTR, and Clec4a4-DTR mice. PCR results are representative of three independent IEC isolations. ( h ) IDO1 protein expression in IECs pooled from three DSS-treated WT or Clec9A DTR mice (day 4). Representative immunoblots for epithelial IDO1 (45 kDa) and β-tubulin control (50 kDa) are shown. ( i ) Absence of CX3CR1 high macrophages does not affect expression of IDO1 and interleukin-18-binding protein (IL-18bp) in IECs during colitis. RT-PCR analysis of Ido1and IL-18bp in IECs obtained from DT-injected untreated or DSS-treated (day 4) WT and CD169-DTR mice. PCR results are representative of three independent IEC isolations.

Article Snippet: Ltd., Singapore) and 3% (w/v) nonfat milk (Bio-Rad) overnight at 4 °C and then incubated for 1 h with the primary antibody rat anti-mouse IDO1 (BioLegend) or polyclonal β-tubulin (Santa Cruz Biotechnology, Dallas, TX) antibody, respectively.

Techniques: Quantitative Proteomics, Biomarker Discovery, Isolation, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Injection, Western Blot, Binding Assay

Journal: Mucosal Immunology

Article Title: Intestinal CD103 + CD11b − dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells

doi: 10.1038/mi.2015.64

Figure Lengend Snippet: IDO1 and IL-18bp expression is modulated by interferon-γ (IFN-γ). ( a ) Colonocytes express IFN-γ receptor (IFN-γR). The ex vivo isolated colonocytes and CMT-93 colon epithelial cell line were analyzed by semiquantitative real-time PCR (RT-PCR) analysis for IFN-γ receptor expression. Hprt was used as an endogenous mRNA control. ( b ) IDO1 and IL-18bp expression is induced by IFN-γ. CMT-93 cells were stimulated overnight with 100 U ml −1 IFN-γ and analyzed for Ido1 and IL-18bp expression by semiquantitative RT-PCR analysis. ( c ) IFN-γ −/− mice do not upregulate Ido1 and IL-18bp epithelial expression upon dextran sodium sulfate (DSS) treatment. Intestinal epithelial cells (IECs) were collected from untreated or DSS-treated wild-type (WT) and IFN-γ −/− mice and evaluated by semiquantitative RT-PCR. One representative sample from each experimental group of three mice is shown. SS, steady state. ( d ) Clec9A–diphtheria toxin receptor (DTR) mice have a decreased proportion of IFN-γ-expressing lamina propria (LP) T cells and intraepithelial lymphocytes (IELs). Representative flow cytometry plots of LP and IELs harvested from wild-type (WT) and Clec9A-DTR mice 4 days after DSS treatment and stained for CD4, CD8, and γ/δ T cell receptor (TCR), respectively (representative fluorescence-activated cell sorting (FACS) dot plot, right panel) and stained for intracellular IFN-γ. Quantification of LP CD4 + T cells, LP CD8 + T cells, LP CD4 − CD8 − T-cell fraction, γ/δ + , and CD8 + IELs expressing IFN-γ. N =6–8 mice pooled from 2 independent experiments±s.e.m. Student's t -test significance: * P >0.01, *** P >0.001, NS, not significant. ( e ) Quantitative real-time PCR (qPCR) analysis of IL-15 , IL-12p40 , IL-12p35 , and IL-13p19 expression in distinct colon dendritic cell (DC) subsets obtained from control WT mice: CD103 + CD11b − , CD103 + CD11b + , and CD103 − CD11b + . Data are representative of 3 independent experiments with 10 mice pooled in each group.

Article Snippet: Ltd., Singapore) and 3% (w/v) nonfat milk (Bio-Rad) overnight at 4 °C and then incubated for 1 h with the primary antibody rat anti-mouse IDO1 (BioLegend) or polyclonal β-tubulin (Santa Cruz Biotechnology, Dallas, TX) antibody, respectively.

Techniques: Expressing, Ex Vivo, Isolation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Control, Flow Cytometry, Staining, Fluorescence, FACS

Journal: Mucosal Immunology

Article Title: Intestinal CD103 + CD11b − dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells

doi: 10.1038/mi.2015.64

Figure Lengend Snippet: Immunostimulatory oligonucleotide (ISS-ODN) treatment limits the colitis severity in Clec9A–diphtheria toxin receptor (DTR) mice. DT-injected wild-type (WT) and Clec9A-DTR mice were injected intraperitoneally (i.p.) 10 μg of ISS-ODN at the start of the dextran sodium sulfate (DSS) treatment (2%) and 4 days later. ( a ) Interferon-γ (IFN-γ) response was measured in the serum collected at day 4. ( b ) Epithelial Ido1 and IL-18bp expression profile at steady-state or under DSS treatment. Representative samples of three WT and Clec9A-DTR mice are shown. ( c ) The body weight was monitored daily over a period of 10 days. Black circles: DT-treated WT control; white circles: DT-treated WT control+ISS-ODN; black squares: DT-treated Clec9A-DTR; white squares: DT-treated Clec9A-DTR+ISS-ODN; Each group: n =6 mice from two independent experiments. Values represent the mean±s.d. ND, not detectable.

Article Snippet: Ltd., Singapore) and 3% (w/v) nonfat milk (Bio-Rad) overnight at 4 °C and then incubated for 1 h with the primary antibody rat anti-mouse IDO1 (BioLegend) or polyclonal β-tubulin (Santa Cruz Biotechnology, Dallas, TX) antibody, respectively.

Techniques: Injection, Expressing, Control

Journal: Oncology Letters

Article Title: Gene silencing of indoleamine 2,3-dioxygenase 1 inhibits lung cancer growth by suppressing T-cell exhaustion

doi: 10.3892/ol.2020.11477

Figure Lengend Snippet: IDO1-siRNA silences IDO1 expression in vitro . (A) IDO1 mRNA expression. LLC cells were transfected with IDO1-siRNA or GL2-siRNA (negative control). Untransfected cells were used as a blank control. IDO1 mRNA expression level was detected by reverse transcription-quantitative PCR (n=3). (B) IDO1 protein expression. Silencing of IDO1 was confirmed by western blotting (n=3) in LLC cells after transfection with IDO1-siRNA, GL2-siRNA or blank control. *P<0.05 and **P<0.01. GL2, glabra 2; IDO1, indoleamine 2,3-dioxygenase 1; siRNA, small interfering RNA.

Article Snippet: The antigens were retrieved using a sodium citrate buffer (0.01M, pH 6.0) in a pressure cooker (100°C) for 15 min and tissue sections were incubated with an IDO1 primary antibody (cat. no. sc-53978; 1:50; Santa Cruz Biotechnology, Inc.) for 16 h at 4°C.

Techniques: Expressing, In Vitro, Transfection, Negative Control, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA

Journal: Oncology Letters

Article Title: Gene silencing of indoleamine 2,3-dioxygenase 1 inhibits lung cancer growth by suppressing T-cell exhaustion

doi: 10.3892/ol.2020.11477

Figure Lengend Snippet: IDO1-siRNA in LLC cells inhibits T-cell exhaustion in vitro . (A and B) LLC cells were transfected with IDO1-siRNA or GL2-siRNA and co-cultured with lymphocytes isolated from spleen. Lymphocytes were collected and (A) PD-1 and (B) BTLA were detected by flow cytometry. n=3. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; GL2, glabra 2; IDO1, indoleamine 2,3-dioxygenase 1; PD-1, programmed death-1; siRNA, small interfering RNA; control, untransfected cells.

Article Snippet: The antigens were retrieved using a sodium citrate buffer (0.01M, pH 6.0) in a pressure cooker (100°C) for 15 min and tissue sections were incubated with an IDO1 primary antibody (cat. no. sc-53978; 1:50; Santa Cruz Biotechnology, Inc.) for 16 h at 4°C.

Techniques: In Vitro, Transfection, Cell Culture, Isolation, Flow Cytometry, Small Interfering RNA, Control

Journal: Oncology Letters

Article Title: Gene silencing of indoleamine 2,3-dioxygenase 1 inhibits lung cancer growth by suppressing T-cell exhaustion

doi: 10.3892/ol.2020.11477

Figure Lengend Snippet: Treatment with IDO1-shRNA inhibits lung cancer growth in vivo . Treatment with IDO1 or scrambled expression vectors was performed by hydrodynamic intravenous tail injection. (A) Tumor tissues were collected from lung cancer-bearing mice after 21 days. Expression of IDO1 in tumors was detected by immunohistochemistry (magnification, ×40). (B) IDO1 expression scores. (C) Time of tumor onset was recorded when the tumor diameter reached 5 mm. (D) Tumor growth curve. (E) Images of tumors. At day 21 after LLC cell inoculation, the mice were sacrificed, and the tumors were measured with a caliper. (F) Tumor weights were measured after 21 days. *P<0.05, **P<0.01 and ***P<0.001. IDO1, indoleamine 2,3-dioxygenase 1; shRNA, short hairpin RNA; control, untreated mice,

Article Snippet: The antigens were retrieved using a sodium citrate buffer (0.01M, pH 6.0) in a pressure cooker (100°C) for 15 min and tissue sections were incubated with an IDO1 primary antibody (cat. no. sc-53978; 1:50; Santa Cruz Biotechnology, Inc.) for 16 h at 4°C.

Techniques: shRNA, In Vivo, Expressing, Injection, Immunohistochemistry, Control

Journal: Oncology Letters

Article Title: Gene silencing of indoleamine 2,3-dioxygenase 1 inhibits lung cancer growth by suppressing T-cell exhaustion

doi: 10.3892/ol.2020.11477

Figure Lengend Snippet: IDO1-shRNA treatment decreases the expression of inhibitory receptors of CD4 + /CD8 + T cells in vivo . Lymphocytes from lymph nodes and spleen of LLC-bearing tumor mice were collected on day 21 injection of LLC cells. CD4 + and CD8 + T cells were stained with anti-PD-1 and anti-BTLA and analyzed by flow cytometry. Expression of PD-1 and BTLA on CD4 + T cells in the (A) lymph nodes and (B) spleen of tumor-bearing mice without treatment, or treated with scrambled-shRNA or IDO1-shRNA. (C) Expression of PD-1 and BTLA on CD8 + T cells from the lymph nodes of tumor-bearing mice without treatment or treated with scrambled-shRNA or IDO1-shRNA. Statistical analysis of the expression of PD1 and BTLA on CD4 + (right panel in A and B) and CD8 + (right panels in C) was performed from three independent experiments. *P<0.05 and **P<0.01. BTLA, B and T lymphocyte attenuator; IDO1, indoleamine 2,3-dioxygenase 1; LN, lymph node; PD-1, programmed death-1; sh, short hairpin; SP, spleen.

Article Snippet: The antigens were retrieved using a sodium citrate buffer (0.01M, pH 6.0) in a pressure cooker (100°C) for 15 min and tissue sections were incubated with an IDO1 primary antibody (cat. no. sc-53978; 1:50; Santa Cruz Biotechnology, Inc.) for 16 h at 4°C.

Techniques: shRNA, Expressing, In Vivo, Injection, Staining, Flow Cytometry

Journal: Oncology Letters

Article Title: Gene silencing of indoleamine 2,3-dioxygenase 1 inhibits lung cancer growth by suppressing T-cell exhaustion

doi: 10.3892/ol.2020.11477

Figure Lengend Snippet: Treatment with IDO1-shRNA in tumor-bearing mice in vivo recovers cytokine secretion. C57BL/6 mice were treated with IDO1-shRNA or scrambled-shRNA by hydrodynamic intravenous tail injection. At day 21, blood from tumor-bearing control and treated mice was collected to detect the serum levels of TNF-α and IL-2 by ELISA. (A and B) Serum TNF-α and IL-2 levels in mice with and without tumors. (C and D) Serum TNF-α and IL-2 levels in the control mice, IDO1-shRNA- and scrambled-shRNA-treated mice. n=3. *P<0.05 and **P<0.01. IL-2, interleukin-2; IDO1, indoleamine 2,3-dioxygenase; shRNA, short hairpin RNA; TNF-α, tumor necrosis factor-α.

Article Snippet: The antigens were retrieved using a sodium citrate buffer (0.01M, pH 6.0) in a pressure cooker (100°C) for 15 min and tissue sections were incubated with an IDO1 primary antibody (cat. no. sc-53978; 1:50; Santa Cruz Biotechnology, Inc.) for 16 h at 4°C.

Techniques: shRNA, In Vivo, Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: 3-hydroxy-L-kynurenamine is an immunomodulatory biogenic amine

doi: 10.1038/s41467-021-24785-3

Figure Lengend Snippet: a Lymphatic collectors from Prox1-cre-ERT2-TdT-LoxP-IDO1 mice, visualized after tamoxifen treatment (+TMX), vs untreated (−TMX) ( n = 24 biologically independent replicates). b Nodal LECs express tdTomato only after tamoxifen treatment (+TMX) ( n = 24 biologically independent replicates). c Thoracic ducts and lymph nodes were harvested from tamoxifen-treated Prox1-Cre-ERT2-TdT-LoxP-Ido1 mice and tamoxifen-treated Prox1-Cre-ERT2-TdT controls ( n = 24 biologically independent replicates). d LECs were sorted from the same mice as in ( c ) using CD31 and TdTomato as markers ( n = 12 biologically independent replicates). e 3-HKA quantification in the lymphatic fluid and plasma ( n = 4 biologically independent samples) of Prox1-Cre-ERT2-TdT-LoxP-Ido1 mice and Prox1-Cre-ERT2-TdT controls treated (+TMX) or untreated (−TMX) with tamoxifen. Data are presented as average ± SD of biological quadruplicates ( n = 4) and analyzed by two-tailed paired student’s t test, p < 0.01 (**). f – i Psoriasis was induced in the Prox1-cre-ERT2-TdT-LoxP-Ido1 as described in Fig. , a more severe psoriasis as indicated by ( f , g , i ) histological evaluation and ( h ) animal survival curve, of tamoxifen-treated mice (+TMX) as compared to the untreated animals (−TMX) ( n = 12 biologically independent samples). i Histological quantification is presented as number of keratin layers, quantified in biological replicates ± SD ( n = 7 biologically independent samples). Data are presented as average ± SEM analyzed by two-tailed paired student’s t test. j Quantitative analysis of pro-inflammatory chemokines and cytokines present in skin lysates from Prox1-cre-ERT2-TdT-LoxP-Ido1 mice (+TMX) and (−TMX) tamoxifen, 4 days after psoriasis induction. Data from independent biological replicates ( n = 4) plotted as mean relative expression ± SD. Significance levels are reported as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) (two-way ANOVA followed by Tukey’s multiple comparison test). k Flow cytometry analysis of lymph node from the same mice as in ( g , h ); representative plot of ICOS + /Ki67 + CD8 + T cells. Bar graph report % of ICOS + /Ki67 + CD8 + T cells for each analyzed mouse and representative plot of CD44 + /CD62L − CD8 + T cells. 3-HKA ( n = 4), imiquimod ( n = 6), or 3-HKA plus imiquimod-treated C57BL/6J mice (n = 4). Data are presented as average ± SEM analyzed by two-tailed unpaired student’s t test, p < 0.001 (***). Source data for ( a – c ) and ( j – k ) are provided as Source data file.

Article Snippet: The vessels were incubated overnight at 4 °C in 1X PBST containing primary antibody IDO1 (ENZO, cat# ALX-804-902-0100, dilution 1:200).

Techniques: Clinical Proteomics, Two Tailed Test, Expressing, Comparison, Flow Cytometry