Journal: Respiratory Research

Article Title: TMPRSS2 is a tumor suppressor and its downregulation promotes antitumor immunity and immunotherapy response in lung adenocarcinoma

doi: 10.1186/s12931-024-02870-7

Figure Lengend Snippet: In vivo and in vitro experimental validation of the bioinformatics findings. TMPRSS2-knockdown tumors display increased tumor-infiltrating lymphocytes, expression of immune checkpoint molecules, and sensitization to immune checkpoint inhibitors. a TMPRSS2 knockdown markedly promoted proliferative and invasive abilities of A549 cells. b TMPRSS2 knockdown increased tumor volume and progression in Lewis tumor mouse models. Lewis tumor cells transfected with ShCon or ShTMPRSS2 lentivirus were subcutaneously injected into mice. The tumor volumes were measured every three days from the fifth day to the fifteenth. Data represent mean ± SEM. SEM, standard error of mean. ShTMPRSS2 versus ShCon group, n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001. c TMPRSS2 knockdown increased MSH6 expression in A549 cells, as evidenced by Western blotting. d TMPRSS2 knockdown enhanced the expression of MHC class I genes ( HLA-A , HLA-B , and HLA-C ) in A549 cells, as evidenced by real-time qPCR. e NK cells co-cultured with TMPRSS2 -knockdown A549 cells showing higher proliferation capacity than NK cells co-cultured with TMPRSS2 -wildtype A549 cells, as evidenced by the EDU proliferation assay. f CD8, CD49b, and PD-L1 immunofluorescence staining in Lewis orthotopic tumors and H-score analysis. ShTMPRSS2 versus shCon group, n = 6 for each group, two-tailed Student’s t test, *** p < 0.001. g - j Comparisons of TNF-α, IFN-γ, PD-1, and LAG3 expression on CD8 + T cells from tumor-infiltrating lymphocytes (TILs) in tumor-bearing mice between TMPRSS2 -knockdown and TMPRSS2 -wildtype group (ShTMPRSS2 versus ShCon group, n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001). TILs were stained with CD3, CD8, TNF-α, and IFN-γ and were then analyzed by flow cytometry. Lymphocytes were gated according to forward scatter and side scatter. CD3 and CD8 staining was used to identify CD8 + T cells. k - m TMPRSS2 -knockdown tumors formed by subcutaneous injection of Lewis cells, as mentioned in ( b ). shCon and shTMPRSS2 tumor-bearing mice were divided into vehicle and BMS-1 groups. The vehicle and BMS-1 groups of mice were treated with solvent and BMS-1, respectively. k Representative images of tumor-bearing mice shown on the left. The right graph showing the change of tumor size in the tumor-bearing mice over time. Data represent mean ± SEM ( n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001); Comparison of the volume ratios of mice tumors after and before treatment with BMS-1 between TMPRSS2 -knockdown and TMPRSS2 -wildtype groups (two-tailed Student’s t test, *** p < 0.001). Comparisons of TNF-α ( l ) and IFN-γ ( m ) expression on CD8 + T cells from TILs in tumor-bearing mice ( n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: After 5 days, when the tumor volume was approximately 4–5 mm3, the mice were randomly divided into six groups, with half of the ShCon and ShTMPRSS2 mice treated with 150 U/L PD1/PDL1 inhibitor BMS-1 (concentration 500 mg/mL; i.p.) (MCE Cat. No. HY-19991) every 3 days.

Techniques: In Vivo, In Vitro, Biomarker Discovery, Knockdown, Expressing, Transfection, Injection, Two Tailed Test, Western Blot, Cell Culture, Proliferation Assay, Immunofluorescence, Staining, Flow Cytometry, Solvent, Comparison

Journal: Frontiers in Immunology

Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

doi: 10.3389/fimmu.2020.598556

Figure Lengend Snippet: PD-1/PD-L1 interaction blockade by Salvia plebeia R. Br. ethanol extract (SPE). Competitive ELISA was performed using PD-1/PD-L1 inhibitor ELISA-screening assay with the indicated concentrations of PD-L1-blocking antibody, SPE, and SPE fractions. (A) Inhibition of the PD-1/PD-L1 interaction by PD-L1-blocking antibody, (B) SPE, and (C) SPE fractions (methylene chloride [MC], ethyl acetate [EA], and n-butanol [BuOH]). Cytotoxicity and T-cell activation by SPE and its ethyl acetate (EA) fraction in vitro . (D) The viabilities of PD-L1 aAPC/CHO-K1 and PD-1 effector cells were assessed using the Cell Counting Kit-8 (CCK) assay after treatment with the indicated concentrations of SPE for 24 h. (E–G) A PD-1/PD-L1 blockade bioassay was performed to assess the inhibitory activities of PD-L1-blocking antibody, SPE, and SPE-EA. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of (E) PD-L1-blocking antibody, (F) SPE, or (G) SPE-EA. Bar graph (mean ± standard error of the mean) statistics were determined with the data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P < 0.001, **P < 0.01, *P < 0.05, n.s, not significant, compared with the control.

Article Snippet: Small molecule PD-L1 inhibitor C1 (BMS-1, # T3655) was purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Screening Assay, Blocking Assay, Inhibition, Activation Assay, In Vitro, Cell Counting, Bioassay, Incubation, Control

Journal: Frontiers in Immunology

Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

doi: 10.3389/fimmu.2020.598556

Figure Lengend Snippet: Cytotoxic and T-cell-mediated killing effect of SPE on colorectal cancer cells. (A) PD-L1 expression on the surface of humanized PD-L1-expressing MC38 cells (hPDL1-MCs) was determined using flow cytometry. (B–D) To measure the immunological activity of SPE, hPDL1-MCs (target cells) were co-cultured with humanized PD-1 mouse splenocytes (B, C) and CD8+ T-cells from the tumor (D) (hPD1-MSs; effector cells) at an effector cell-to-target cell ratio of 5:1 and were treated with SPE or 50% DMSO (control). (B) After 72 h of incubation, surviving hPD1-MSs in a 96-well plate underwent the CCK assay to assess cell viability. (C) Co-cultured CellTrace™ Far Red-labeled hPDL1-MCs were treated with SPE (25 μg/mL) or 50% DMSO (control), and the findings were compared with those in the absence of effector cells. The images were obtained under a fluorescence microscope. (D) The cytotoxicity of CD8+ T-cells against hPD-L1-MCs at an effector cell-to-target cell ratio of 5:1 was analyzed using an LDH assay kit (percentages calculated according to kit instruction). ***P < 0.001, compared with the control.

Article Snippet: Small molecule PD-L1 inhibitor C1 (BMS-1, # T3655) was purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Control, Incubation, Labeling, Fluorescence, Microscopy, Lactate Dehydrogenase Assay

Journal: Frontiers in Immunology

Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

doi: 10.3389/fimmu.2020.598556

Figure Lengend Snippet: PD-1/PD-L1 interaction blockade and T-cell activation enhancement by SPE components in vitro . (A) Chemical components of SPE. (B) The ability of seven components from SPE to enhance T-cell functional activity was assessed using a PD-1/PD-L1 blockade bioassay. These seven components were used at a concentration of 2 µM. (C, D) A PD-1/PD-L1 blockade bioassay was performed to measure the inhibitory activity of apigenin and cosmosiin. PD-L1 aAPC/CHO-K1 cells were plated and incubated at 37°C for 20 h prior to the addition of PD-1 effector cells and increasing concentrations of apigenin or cosmosiin. (E, F) Competitive ELISA was performed using a PD-1/PD-L1 inhibitor ELISA-binding assay after treatment with the indicated concentrations of (E) apigenin and (F) cosmosiin. Bar graph (mean ± standard error of the mean) statistics were determined with data of three experiments, using one-way ANOVA with Tukey’s post-hoc test; ***P < 0.001, **P < 0.01, *P < 0.05, n.s, not significant, compared with the control.

Article Snippet: Small molecule PD-L1 inhibitor C1 (BMS-1, # T3655) was purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Activation Assay, In Vitro, Functional Assay, Activity Assay, Bioassay, Concentration Assay, Incubation, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Binding Assay, Control

Journal: Frontiers in Immunology

Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

doi: 10.3389/fimmu.2020.598556

Figure Lengend Snippet: Global kinetic analysis of cosmosiin binding to biotinylated (A) PD-1 and (B) PD-L1 immobilized on a streptavidin biolayer interferometry (BLI) sensor. The kinetics of cosmosiin for immobilized PD-1 and PD-L1 were monitored with increasing concentration of cosmosiin (0, 0.125, 0.25, and 0.375 mM), dissolved in PBS buffer (pH 7.3) containing 1% DMSO.

Article Snippet: Small molecule PD-L1 inhibitor C1 (BMS-1, # T3655) was purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Binding Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

doi: 10.3389/fimmu.2020.598556

Figure Lengend Snippet: Protein–ligand docking simulation between PD-L1 (A) /PD-1 (B) and the Salvia plebeia R. Br. ethanol extract (SPE) component cosmosiin. Binding models were obtained through docking simulation of cosmosiin on PD-L1 and PD-1 derived from the PD-1/PD-L1 complex (PDB code 4ZQK) using AutoDock Vina. The hydrogen bonds and hydrophobic interactions between PD-L1/PD-1 and cosmosiin were analyzed using LigPlot+.

Article Snippet: Small molecule PD-L1 inhibitor C1 (BMS-1, # T3655) was purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Binding Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model

doi: 10.3389/fimmu.2020.598556

Figure Lengend Snippet: Anti-tumor efficacy of SPE on hPD-L1 MC38 tumor growth in hPD-1 knock-in mice. (A) Representative images show the tumor development at 16 days after treatment. Mice were inoculated s.c. with hPD-L1 MC38 cells and were administrated with aPD-1 (5 mg/kg, i.p.) or SPE (100, 300 mg/kg, i.g.) (n=3). Tumor mice received an equivalent volume of vehicle (200 µL). (B) Image of subcutaneous tumor mass on day 16 (scale bars, 1 cm) (n=3). (C) Mean tumor weight (n=3). (D) Mean tumor diameter (n=3). (E) Immunohistochemistry staining of CD3+ in humanized PD-1 mouse model of tumor. (F) FACS analysis of tumor CD8+ T-cell populations from human PD-L1 knock in MC 38 tumor-bearing model of humanized PD-1 mice. Percentages of polyclonal CD8+ T-cells of CD45+ cells in tumor. Bar graph (mean ± standard error of the mean) statistics were determined with one-way ANOVA with Tukey’s post-hoc test; ***P < 0.001, **P < 0.01, *P < 0.05, compared with the vehicle group.

Article Snippet: Small molecule PD-L1 inhibitor C1 (BMS-1, # T3655) was purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: Knock-In, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic illustration of US-triggered deep-tissue activation of SPINs to release immunomodulators. b Schematic illustration of sonodynamic activation of SPINs to debulk tumor, enhance tumor immunogenicity, and release immunomodulators in situ as well as synergetic action of IDO inhibition and PD-L1 blocking on enhancing antitumor immunity with alleviated irAEs relative to free-drug administration.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Activation Assay, In Situ, Inhibition, Blocking Assay

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Chemical structures of amphiphilic semiconducting polymeric modulators and schematic illustration of their self-assembly and surface modification to form SPINs. b The molar ratios of each component in different SPINs. c Zeta potentials and hydrodynamic sizes of different SPINs in 1× PBS buffer (pH = 7.4) ( n = 4). d Photographs of erythrocytes after incubation with 1× PBS buffer (negative control), 1% Triton X-100 (positive control), and 1× PBS buffer containing SPINs at the concentration of 100 µg/mL for 2 h, followed by centrifugation. e Hemolysis percentages of erythrocytes after incubation with SPINs at different concentrations for 2 h ( n = 4). f Schematic illustration of US irradiation of SPIN D2 solutions covered with a pork tissue. g ESR spectra of 1 O 2 for SPIN D2 (20 µg/mL) after US irradiation (1.2 W/cm 2 , 3 min) without or with coverage of pork tissues at different thicknesses. h Release profiles of aPD-L1 and NLG919 from SPIN D2 (40 µg/mL) after US irradiation for different time ( n = 4). i PD-L1/PD-1 binding activity assay after treatment with free aPD-L1 or SPIN D2 (40 µg/mL) with or without US irradiation ( n = 4). SPIN D2 – US versus SPIN D2 + US: P < 0.0001. Statistical significance was calculated via a two-tailed Student’s t test. *** P < 0.001. In ( g – i ), the power intensity of US irradiation was 1.2 W/cm 2 (1.0 MHz, 50% duty cycle). Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Modification, Incubation, Negative Control, Positive Control, Concentration Assay, Centrifugation, Irradiation, Binding Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schedule for the establishment of primary and distant tumors, triple systemic injection of SPINs (0.2 mL, 0.6 mg/mL) via tail vein, US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min), and analysis of immune responses. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 6) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). SPIN D2 + US versus drug + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus drug: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) receiving different treatments as indicated. e Schematic illustration of treatment of rechallenged tumor mouse models using SPINs. f Growths of rechallenged tumors in Panc02 tumor-bearing mice after injection of saline or SPIN D2 (0.2 mL, 0.6 mg/mL) with US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min) ( n = 5). Saline versus SPIN D2 + US: P < 0.0001. g The survival curves of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 10). h Flow cytometry analysis of populations of effector memory T cells in the spleen of Panc02 tumor-bearing C57BL/6 mice after different treatments followed by tumor rechallenge ( n = 4). Saline versus SPIN D2 + US: P = 0.0059. i Differentially expressed gene numbers in tumor tissues of mice after different treatments. j Relative expression of Carl , Hmgb1-ps1 , Hmgb1-ps2 , Cd80 , Cd86 , Cd40 , Pdcd1 , Cd3e , Cd8a , Ifng , Gzmb , Cxcl1 , Cxcl2 , Cxcl9 , Cxcl10 , Cxcl11 , Ccl4 , Ccl5 , Il1b , Il2 , Il6 , Il7 , Il15 , Ido1 , and Cd274 in tumors of Panc02 tumor-bearing mice after different treatments (the experiment was repeated independently five times with similar results). k Unsupervised hierarchical clustering of relative gene expression in tumors of Panc02 tumor-bearing C57BL/6 mice after different treatments ( n = 5). Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a Schematic of sono-immunotherapy of subcutaneous pancreatic mouse tumors covered with 5-cm tissue. b , c Relative tumor volumes of primary ( b ) and distant ( c ) tumors of Panc02 tumor-bearing C57BL/6 mice ( n = 5) after systemic injection of saline, free-drug mixture (on day 0, 3, and 6, 4 mg/kg body weight for NLG919 and aPD-L1), SPIN 0 or SPIN D2 (0.2 mL, 0.6 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). The primary tumors were covered with 5-cm tissue under US irradiation. SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for primary tumors ( b ); SPIN D2 + US versus SPIN 0 + US: P < 0.0001 for distant tumors ( c ). d Survival curves of Panc02 tumor-bearing C57BL/6 mice ( n = 10) after different treatments for 60 days. e Schematic of US-mediated deep-tissue sonodynamic therapy of orthotopic pancreatic rabbit tumors. f Radiolabeling stability of 131 I-SPIN 0 after storage in saline or 50% serum at 37 °C for different time ( n = 3). g , h SPECT imaging ( g ) and signal intensity ( h ) of orthotopic pancreatic rabbit tumors after systemic injection of 131 I-SPIN 0 (1.0 mL, 1.5 mg/mL) for different time ( n = 4). The white dotted circle indicated tumors. i Computed tomography (CT) imaging of orthotopic pancreatic rabbit tumors after systemic injection of saline or SPIN 0 (1.0 mL, 1.5 mg/mL) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 30 min). The white dotted circle indicated tumors. j Tumor volume of orthotopic pancreatic rabbit tumors ( n = 3) after treatments as indicated for different days. Saline + US versus SPIN 0 + US: P = 0.0108. k H&E staining images of orthotopic pancreatic rabbit tumors after different treatments. The experiment was repeated independently three times with similar results. l Survival curves of orthotopic pancreatic tumor-bearing rabbits ( n = 4) after different treatments for 20 days. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; * P < 0.05, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Injection, Irradiation, Radioactivity, Single Photon Emission Computed Tomography, Imaging, Computed Tomography, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Precision cancer sono-immunotherapy using deep-tissue activatable semiconducting polymer immunomodulatory nanoparticles

doi: 10.1038/s41467-022-31551-6

Figure Lengend Snippet: a , b Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( a ), and CD3 + CD8 + CTLs ( b ) in blood of mice ( n = 4) at 30 day after systemic administrations of saline, SPIN 0 , SPIN D2 (0.2 mL, 1.2 mg/mL) or free-drug mixture (8 mg/kg body weight for NLG919 and aPD-L1) with or without US irradiation (1.0 MHz, 1.2 W/cm 2 , 50% duty cycle, 10 min). Saline – US versus drug − US: P = 0.0023; saline − US versus drug + US: P = 0.0006; drug + US versus SPIN D2 + US: P = 0.0071 for CD3 + CD4 + Th cells ( a ); saline − US versus drug − US: P = 0.0004; saline − US versus drug + US: P = 0.0001; drug + US versus SPIN D2 + US: P = 0.0093 for CD3 + CD8 + CTLs ( b ). c , d Flow cytometry analysis of percentages of CD3 + CD4 + Th cells ( c ), and CD3 + CD8 + CTLs ( d ) in spleen of mice ( n = 4) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0008; saline − US versus drug + US: P = 0.0005; drug + US versus SPIN D2 + US: P = 0.0015 for CD3 + CD4 + Th cells ( c ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P = 0.0002; drug + US versus SPIN D2 + US: P = 0.0049 for CD3 + CD8 + CTLs ( d ). e Representative H&E staining images of liver after 30 days of treatments in different groups (white arrows indicate the infiltrated lymphocytes). The experiments were repeated independently three times with similar results. f Heatmap to show relative fold of cytokine levels in serum of mice after different treatments for 30 days relative to those in saline control group. g , h Serum levels of ALT ( g ) and AST ( h ) in mice ( n = 5) after different treatments for 30 days. Saline − US versus drug − US: P = 0.0010; saline − US versus drug + US: P = 0.0020; drug + US versus SPIN D2 + US: P = 0.0054 for ALT ( g ); saline − US versus drug − US: P = 0.0001; saline − US versus drug + US: P < 0.0001; drug + US versus SPIN D2 + US: P = 0.0013 for AST ( h ). i Summary comparison of the antitumor immunity and irAEs between SPIN D2 -mediated sono-immunotherapy and free-drug treatment. Data are presented as mean values ± SD. Statistical significance was calculated via two-tailed Student’s t test; ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Mouse PD-1[biotinylated]: PD-L1 inhibitor screening assay kit was purchased from BPS Bioscience (San Diego, CA, USA).

Techniques: Flow Cytometry, Irradiation, Staining, Two Tailed Test