Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Strategy and materials in this study. (A) Diagram of the procedure in the study. The primary tumor cells and tumor organoids were generated from patients' tumor tissues and ROBO1-NK were constructed from patients' PBMCs. (B) IHC of ovarian tumor tissues. Negative control was used irrelevant IgG replacing primary antibody for incubating. Strong positive of ROBO1 expression on cell membrane was marked by red circles. (C) ROBO1 positively expressed in tumor tissues of the clinical ovarian cancer patient #01, #02, #03 and #04.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Generated, Construct, Negative Control, Expressing, Membrane

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: Characters of ROBO1-NK cells. (A) Morphology comparison of PBMCs (upper panel), PBMC-NK cells (mid panel) and ROBO1-NK cells (bottom panel) under bright field. (B) Biomarkers determination of ROBO1-NK cells by flow cytometry. For the biomarkers CD34, CD45, CD3, CD56 and CD16, negative controls were used irrelevant IgG replacing primary antibody for incubating, presented as red peaks. For the ROBO1-CAR detection, negative control was used PBMC-NK incubating with anti-CAR antibody, presented as red peak. Samples in detection were presented as blue peaks.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Comparison, Flow Cytometry, Negative Control

Journal: Future Science OA

Article Title: Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids

doi: 10.2144/fsoa-2023-0135

Figure Lengend Snippet: ROBO1-NK lyse ovarian cancer cell line and primary cancer cells. (A) Positive expression of ROBO1 on SKOV-3 cells. (B) Rate of SKOV-3 cell lysis with different E-T ratio. E: number of effective cells; T: number of target cells. (C) Time curve of different NK cells, including Mock-CAR-NK (blue), PBMC-NK (green), ROBO1-NK (red) and control (medium, black), lysing SKOV-3 cells. (D) Lysis rate of primary ovarian tumor cells from patient #01, #02, #03 and #04 by PBMC-NK and ROBO1-NK after 24 h, detected with CCK-8 assay. (E) Photograph of PBMC-NK and ROBO1-NK lysing efficacy on primary ovarian tumor cells after 48 h co-culturing. The primary cells were presented as spindle-like form while NK cells were spherical form with clusters.

Article Snippet: In brief, PBMCs were isolated from the patient's peripheral blood using the MACSprepTM PBMC Isolation Kit (130-115-169, Miltenyi Biotec, Bergisch Gladbach, GER) employing the density gradient centrifugation method.

Techniques: Expressing, Lysis, Control, CCK-8 Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: Effect of PEG-Liker (PEG-IALLIPF), Trp, or MPP-Trp on the production of NO and Pro-inflammatory cytokines. ( A ) In Control-PBMCs, Asthma-PBMCs, Asthma-PBMCs + PEG-Liker, Asthma-PBMCs + Trp and Asthma-PBMCs + MPP-Trp group, NO production were examined by NO assay kit. Pro-inflammatory cytokines ( B ) TNF-α, ( C ) IL-1β, and ( D ) IL-6 contents in all groups were examined by ELISA assay. Data were presented as mean ± SD of three independent experiments. * P <0.05, ** P <0.01, *** P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: MPP-Trp was closely correlated with the balanced Th1/Th2 level and Th1/Th2-type cytokine production. ( A ) In Control-PBMCs, Asthma-PBMCs, Asthma-PBMCs + PEG-Liker, Asthma-PBMCs + Trp and Asthma-PBMCs + MPP-Trp group, Th1 population (CD4+IFN-γ+) and Th2 population (CD4+IL-4+) were selected by flow cytometry assay. ( B ) Relative mRNA expressions of IFN-γ, IL-4, IL-13, and IL-5 in all groups were assessed by qRT-PCR. ( C ) The IFN-γ, IL-4, IL-13, and IL-5 contents in all groups were examined by ELISA assay. Data were presented as mean ± SD of three independent experiments. * P <0.05, ** P <0.01, *** P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Control, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: MPP-Trp altered cytokine gene expression and production in a concentration-dependent way. In Control-PBMCs, Asthma-PBMCs, and Asthma-PBMCs + MPP-Trp (10, 50, 100 and 200 μg/mL) group, ( A ) Th1/Th2 cytokine gene expressions (IFN-γ, IL-4, IL-13, and IL-5) were determined by RT-qPCR. ( B ) The cytokines (IFN-γ, IL-4, IL-13, and IL-5) levels in all groups were detected by ELISA. Data were presented as mean ± SD of three independent experiments. ** P <0.01, *** P <0.001 vs Control-PBMCs group; # P <0.05, ## P <0.01, ### P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Gene Expression, Concentration Assay, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Cathepsin B-Activatable Bioactive Peptide Nanocarrier for High-Efficiency Immunotherapy of Asthma

doi: 10.2147/IJN.S455633

Figure Lengend Snippet: MPP-Trp altered cytokine gene expression and production in a time-dependent way. ( A ) In Control-PBMCs, Asthma-PBMCs, and 100 μg/mL Asthma-PBMCs + MPP-Trp (6, 12, 24, and 48 h) group, IFN-γ, IL-4, IL-13, and IL-5 mRNA levels were determined by RT-qPCR. ( B ) The Th1/Th2-type cytokines (IFN-γ, IL-4, IL-13, and IL-5) productions in all group were examined by ELISA. Data were presented as mean ± SD of three independent experiments. *** P <0.001 vs Control-PBMCs group; # P <0.05; ## P <0.01; ### P <0.001 vs Asthma-PBMCs group.

Article Snippet: The total peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples of control group and asthma group by mouse peripheral blood mononuclear cell isolation kit (P6340; Solarbio, Beijing, China) according to the manufacturer's protocol.

Techniques: Gene Expression, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: NAR Molecular Medicine

Article Title: IgAim: cell surface Ig-aimed immune memory erasers for the therapy of autoimmune diseases and B leukemia

doi: 10.1093/narmme/ugaf016

Figure Lengend Snippet: Selective removal of B cells from mixed cells using IgAim. ( A ) Three different classes of B-cell lines, namely sIgM + B-cell line Ramos, sIgA + B-cell line U-2904, and sIgG + B-cell line anti-P7 Raji, were mixed in a 1:2:2 ratio considering the proliferation rates of each cell type. These mixed cell lines were cultured for 3 days in the presence of 1 μg/ml IgAimG, IgAimA, and IgAimP7. The proportions of each cell line were analyzed by the flow cytometry. ( B ) Lymphocytes were identified from PBMCs based on morphological parameters, and primary B cells were then gated based on CD19 expression. ( C ) PBMCs were cultured for 3 days in the presence of 1 μg/ml IgAimG and IgAimA, and the proportions of sIgG + and sIgA + primary B cells were analyzed by the flow cytometry.

Article Snippet: Primary peripheral blood mononuclear cells (PBMCs) [ATCC (LGC Standards S.r.L.), PCS-800–011] were washed once with RPMI medium containing 20% FBS and then cultured at 37°C for 1 h in a Falcon tube in 10 ml of RPMI medium containing 150 U of DNase I to degrade DNA released from dead cells that can cause cell clumps.

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity

doi: 10.1101/2022.04.12.487988

Figure Lengend Snippet: (a-e) Human PBMC from donors with acute SARS-CoV-2 infection were stimulated with SARS-CoV-2 peptide pools (Spike and Membrane, Mem) and treated with Avasimibe (AVS) or DMSO for 8d. SARS-CoV-2-specific cytokine production by CD4 + T cells was detected via flow cytometry. The cytokine production/CD154 expression in wells without peptide stimulation was subtracted to determine SARS-CoV-2-specific cytokine production/CD154 expression in summary data. Example plots and summary data for SARS-CoV-2 specific IFNγ( a ), TNF ( b ), MIP1β( c ) production and CD154 expression ( d ) by CD4 + T cells (n=19). ( e ) Assessment of SARS-CoV-2-specific proliferation determined by CFSE dilution gated on IFNγ + CD4 + T cells (Spike n=10; Mem n=11). Bars mean. Doughnut charts indicate fraction of donors with response to AVS (red). Response defined as de novo or increased cytokine production/CD154 expression. P values determined by Wilcoxon matched-pairs signed rank test.

Article Snippet: PBMC were expanded at 37°C for 8d±0.5μM of the ACAT inhibitor Avasimibe (AVS; Selleckchem) or equivalent concentration of DMSO replenished every 2d.

Techniques: Infection, Membrane, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

doi: 10.3389/fimmu.2021.627020

Figure Lengend Snippet: Growth of CLL B cells only occurs when there is associated expansion of autologous T cells. Representative × 10 original magnification IH images of splenic tissue obtained at euthanasia (A) Few CD20 + cells with no CD3 + cells or Ki67 + cells are present (CLL1083, representative result from 10/15 animals). (B) Aggregates of CD20 + and CD3 + cells with Ki67 + cells are apparent around blood vessels (perivascular aggregates, PVAs) (CLL1279, representative of 5/15 animals).

Article Snippet: At the end of the T-cell activation period, autologous CLL PBMCs from the same patient (all experiments) were thawed and evaluated by trypan blue (Thermo Fischer Scientific).

Techniques:

Journal: Frontiers in Immunology

Article Title: A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

doi: 10.3389/fimmu.2021.627020

Figure Lengend Snippet: Co-injection of autologous activated T cells with CLL PBMCs leads to more effective engraftment and growth in NSG mice. Time course quantification by FC of human CD45 + cells, CLL B cells and autologous CLL T cells from single cell suspensions in the peripheral blood, spleen and bone marrow of 4 different patients evaluated independently. PBMCs with or without aT were injected into 5 mice per group and bled weekly up to euthanasia at week 4 (n = 40 mice); for two of the patients, additional groups of mice were injected, and these were euthanized at weeks 5, 7 and 9. Points represent the median and the interquartile range. * corresponds to Mann-Whitney U test P values < 0.05, ** P < 0.01, and *** P < 0.001. On the right, spleens of 10 mice at week 5 post injection of 20 × 10 6 CLL PBMCs with (Left; n = 5) and without (Right; n = 5) 0.5 × 10 6 activated T cells (aT).

Article Snippet: At the end of the T-cell activation period, autologous CLL PBMCs from the same patient (all experiments) were thawed and evaluated by trypan blue (Thermo Fischer Scientific).

Techniques: Injection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

doi: 10.3389/fimmu.2021.627020

Figure Lengend Snippet: Busulfan preconditioning does not provide a clear advantage for the xenografting of primary CLL cells in the PBMC + aT model. (A) Five NSG mice did not or did receive 25mg/kg busulfan ip 24 h prior to xenografting. Then, 20 × 10 6 CLL PBMCs with 0.5 × 10 6 activated T cells (aT) were injected iv into NSG mice. Five weeks after cell injection, mice were sacrificed and single cell suspensions from blood, spleen, bone marrow (BM) and peritoneum were analyzed by flow cytometry. Busulfan did not significantly improve CLL B-cell (top) and T-cell (bottom) engraftment. Data represent a composite of experiments involving cells from 4 patients, 2 U-CLL and 2 M-CLL. (B) Similar busulfan preconditioning was given or not to two other sets of 5 NSG mice that received samples from 4 different patients (2 U-CLL and 2 M-CLL). Twenty-four h after, 20 × 10 6 CLL PBMCs with 0.5 × 10 6 activated T cells (aT) were injected ip into each recipient mouse. Although there is a trend for better engraftment of CLL B and T cells in busulfan-pretreated mice, there are no significant differences between the numbers of CLL B and T cells in any of the groups. Bar graphs represent the mean fold change (after setting the average cell counts obtained from PBMC mouse spleens as 1); S.E.M. determined by Mann-Whitney U test. n/s: no statistically significant difference.

Article Snippet: At the end of the T-cell activation period, autologous CLL PBMCs from the same patient (all experiments) were thawed and evaluated by trypan blue (Thermo Fischer Scientific).

Techniques: Injection, Flow Cytometry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

doi: 10.3389/fimmu.2021.627020

Figure Lengend Snippet: T-cell findings in the PBMC + aT PDX model. (A) T cells residing in NSG spleens following transfer are principally CD4 + . Top. The percentage of CD5 + CD4 + as a total of all T cells obtained by flow cytometry analysis of spleen at euthanasia at the time points shown. Data obtained from transfer of cells into 25 mice with euthanasia of 5 animals at each time point. Median percentage CD5 + CD4 + cells indicated by bar and percentage figure. Bottom. The percentage of CD5 + CD4 + as a total of all T cells obtained by flow cytometry analysis of spleen at euthanasia. Data obtained from 13 independent experiments (each corresponding to a different CLL case number) where transfer had been made at least 28 days earlier. Median percentage CD5 + CD4 + cells indicated by bar and percentage figure. n/s: no statistically significant differences. (B) Representative FC and IH findings of CD4 + and CD8 + staining in spleen. Images at × 10 original magnification. Pale central areas correspond to CD20 + PVAs. For CLL1279, CD4 + cells are located especially around the rim of the known location of CD20 cells; a minimal number of CD8 + cells are present. CLL1623 has both CD4 + and CD8 + cells; CD4 + cells again locate around and within CD20 + aggregates. In CLL1083, CD4 + cells are densely present within the CD20 + PVAs with less at the outer margin.

Article Snippet: At the end of the T-cell activation period, autologous CLL PBMCs from the same patient (all experiments) were thawed and evaluated by trypan blue (Thermo Fischer Scientific).

Techniques: Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

doi: 10.3389/fimmu.2021.627020

Figure Lengend Snippet: IP administration of CLL B and T cells gives rise to different distributions and activation states. (A) Time course quantification of human CD45-expressing cells, CLL B cells and human T cells evaluated by FC in the peripheral blood of mice injected with 20 × 10 6 CLL PBMCs with 0.5 × 10 6 activated T cells either iv or ip (4 patients’ samples, 5 mice per patient and condition). Points represent the median and the interquartile range. (B) CLL B and T cell absolute counts at day 28 post injection in peripheral blood, spleen, peritoneum and bone marrow from mice injected iv (n = 20) or ip (n = 20). (C) Percentage of divided CLL B cells evaluated by CFSE dilution by FC in the spleen and the peritoneum over time. * corresponds to Mann-Whitney U test P values < 0.05, ** P < 0.01, and *** P < 0.001. n/s: no statistically significant difference.

Article Snippet: At the end of the T-cell activation period, autologous CLL PBMCs from the same patient (all experiments) were thawed and evaluated by trypan blue (Thermo Fischer Scientific).

Techniques: Activation Assay, Expressing, Injection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

doi: 10.3389/fimmu.2021.627020

Figure Lengend Snippet: The PBMC + aT PDX model demonstrates the activity of a CD19xTcR-specific DART. (A) Mice (n = 15) were injected with 0.5 × 10 6 anti-CD3/28 + IL-2 pre-activated CLL-derived T cells and 20 × 10 6 CLL PBMCs on Day 0. Expansion of both T and B cells was determined 25 days post transfer by detection of human IFNγ, and human IgG in murine plasma samples. DART bispecific antibodies (DART molecule, n = 4 animals; or DART control molecule, n = 4 animals) or saline were administered ip from Day 30 to Day 34. Plasma levels of human IFNγ and IgG were further determined at day 46 when euthanasia was performed. Animals with <10 pg/ml IFNγ at Day 25 were excluded from receiving DARTs and were not included in the analysis making 3 groups of 4 animals for each condition. (B) Plasma levels of IFNγ and IgG in the 3 groups of animals (n = 12, 4 per group), taken pre-DART injection at Day 25 and at euthanasia at Day 40. Results show no significant differences in IFNγ levels between the 3 groups before DART molecule injection and at euthanasia. In contrast, plasma Ig levels are significantly lower in CD19xTcR DART-treated animals (red dots), compared to the DART control molecule-treated control animals (blue dots). * corresponds to Kruskal-Wallis test P value < 0.05. (C) FC reveals absence of CD5 + CD19 + cells in DART molecule-treated animals. Illustrative FC plots are representative of hCD45 single cell splenic suspensions obtained at euthanasia. Graph shows median CD5 + CD19 + cells in each group as a percentage of total hCD45 + cells isolated. Compared to DART control molecule-treated control animals (blue dots), there are significantly fewer CD5 + CD19 + cells in DART molecule-treated animals (red dots). * corresponds to Kruskal-Wallis test P value < 0.05. (D) Representative IHC (x20 original magnification) of DART control molecule-treated animals (upper panel) compared to DART molecule-treated animals (lower panel). These results were apparent for both a U-CLL1539 (illustrated in Figure) and M-CLL0545.

Article Snippet: At the end of the T-cell activation period, autologous CLL PBMCs from the same patient (all experiments) were thawed and evaluated by trypan blue (Thermo Fischer Scientific).

Techniques: Activity Assay, Injection, Derivative Assay, Clinical Proteomics, Control, Saline, Isolation

Journal: Parasites & Vectors

Article Title: A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

doi: 10.1186/s13071-018-2745-4

Figure Lengend Snippet: Fasciola gigantica -derived rFg14-3-3e protein binds to the surface of goat PBMCs. Visualization of rFg14-3-3e protein attachment to goat PBMCs surface was carried out by incubation of goat PBMCs treated or untreated with rFg14-3-3e protein with rat anti-rFg14-3-3e primary antibody. Hoechst (blue) and Cy3-conjugated secondary antibody (red) were used to stain host cell nuclei and rFg14-3-3e protein, respectively. Surface staining was detected in rFg14-3-3e-treated cells. No staining was detectable in untreated cells. Scale-bars : 10 μm

Article Snippet: Anti-proliferative effects of rFg14-3-3e protein on goat PBMCs were determined by performing Cell Counting Kit-8 (CCK-8) assay (Beyotime, Haimen, Jiangsu, China).

Techniques: Derivative Assay, Incubation, Staining

Journal: Parasites & Vectors

Article Title: A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

doi: 10.1186/s13071-018-2745-4

Figure Lengend Snippet: rFg14-3-3e protein induced polarized patterns of cytokine secretion. Goat PBMCs were incubated for 24 h in the presence or absence of serial concentrations of rFg14-3-3e protein. The levels of cytokine concentration in the supernatant of cultured goat PBMCs was quantified by ELISA. Graphs represent means ± standard deviations of data from 3 independent biological replicates. Asterisks indicate statistical significance between treated and untreated control goat PBMCs (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, non-significant)

Article Snippet: Anti-proliferative effects of rFg14-3-3e protein on goat PBMCs were determined by performing Cell Counting Kit-8 (CCK-8) assay (Beyotime, Haimen, Jiangsu, China).

Techniques: Incubation, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Parasites & Vectors

Article Title: A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

doi: 10.1186/s13071-018-2745-4

Figure Lengend Snippet: rFg14-3-3e protein inhibited goat PBMCs proliferation. Goat PBMCs were sham-treated with control buffer or with serial concentrations of rFg14-3-3e protein and incubated for 72 h at 37 °C at 5% CO 2 . Proliferation of cells was determined using CCK-8 assay. Results indicate that rFg14-3-3e protein significantly inhibited goat PBMCs proliferation. Graphs represent means ± standard deviations of data from 3 independent biological replicates. Asterisks indicate significance difference between treated cells and control cells (**** P < 0.0001)

Article Snippet: Anti-proliferative effects of rFg14-3-3e protein on goat PBMCs were determined by performing Cell Counting Kit-8 (CCK-8) assay (Beyotime, Haimen, Jiangsu, China).

Techniques: Control, Incubation, CCK-8 Assay

Journal: Parasites & Vectors

Article Title: A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

doi: 10.1186/s13071-018-2745-4

Figure Lengend Snippet: rFg14-3-3e protein suppressed goat PBMCs migration. Goat PBMCs were sham-treated with control buffer or with serial concentrations of rFg14-3-3e protein, then the cell migration percentage (%) was determined. Graphs represent means ± standard deviations of data from 3 independent biological replicates. The asterisks indicate significant difference between treated and sham-treated control cells (** P < 0.001; **** P < 0.0001; ns, non-significant)

Article Snippet: Anti-proliferative effects of rFg14-3-3e protein on goat PBMCs were determined by performing Cell Counting Kit-8 (CCK-8) assay (Beyotime, Haimen, Jiangsu, China).

Techniques: Migration, Control

Journal: Parasites & Vectors

Article Title: A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

doi: 10.1186/s13071-018-2745-4

Figure Lengend Snippet: Effects of rFg14-3-3e protein on intracellular NO production. Goat PBMCs were sham-treated with control buffer or with serial concentrations of rFg14-3-3e protein and maintained at 37 °C. NO concentration in the goat PBMCs was measured by Griess assay. Graphs represent means ± standard deviations of data from 3 independent biological replicates. Asterisks indicate significant difference between treated and non-treated control cells (** P < 0.01; ns, non-significant). The inhibitory effect was only statistically significant at the highest concentration (80 μm/ml)

Article Snippet: Anti-proliferative effects of rFg14-3-3e protein on goat PBMCs were determined by performing Cell Counting Kit-8 (CCK-8) assay (Beyotime, Haimen, Jiangsu, China).

Techniques: Control, Concentration Assay, Griess Assay

Journal: Parasites & Vectors

Article Title: A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

doi: 10.1186/s13071-018-2745-4

Figure Lengend Snippet: rFg14-3-3e protein induced apoptosis in goat PBMCs. Apoptotic cells were determined by Annexin V/PI staining and flow cytometry analysis. a Dot plot showing death of goat PBMCs in response to exposure to rFg14-3-3e protein. b Apoptotic cells (Annexin V+/PI-) were plotted and compared with percentage of cell population. Graphs represent means ± standard deviations of data from 3 independent biological replicates. The asterisks indicate significant differences between treated and untreated control goat PBMCs (**** P < 0.0001)

Article Snippet: Anti-proliferative effects of rFg14-3-3e protein on goat PBMCs were determined by performing Cell Counting Kit-8 (CCK-8) assay (Beyotime, Haimen, Jiangsu, China).

Techniques: Staining, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin

doi: 10.1038/srep29736

Figure Lengend Snippet: ( a ) Proportion (%) of circulating B cells positive for the skin homing marker CLA from patients with melanoma (n = 49) and healthy volunteers (n = 24), quantified by multi-color flow cytometric evaluations. Peripheral blood B cells were identified as CD3-CD14-CD19+CCD22+ cells (top panel). Representative dot plots of CLA+CD45+B cells from a melanoma patient and a healthy volunteer are shown (bottom panel). Quantification was based on % CLA+B cells from CD45+CD3-CD14-CD19+CCD22+PBMCs (right panel). ( b ) CD22+ cells in normal skin (left), cutaneous melanoma lesions (middle) and lymph node metastases (right) were detected by immunohistochemistry (tissue microarrays, top/bottom: example images per cohort, acquired on a Leica AxioScan, 40x objective; Scale bars: 66 μm). ( c ) CD22+ cell infiltrates per high-powered field (HPF) were quantified; Top panel: n = 189 melanomas, n = 16 normal skin samples; Bottom panel: CD22+ infiltrates stratified into cutaneous, lymph node and distant metastases. ( d ) B cells were detected from peripheral blood, melanoma lesion and normal skin samples using antibodies against CD19+, CD22+ (both FITC) and CD45+ (PE) and analysed by flow cytometry (example from matched blood, normal skin and cutaneous melanoma samples derived from a patient with melanoma).

Article Snippet: For evaluations of CLA+B cells, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque Plus density centrifugation GE Healthcare).

Techniques: Marker, Immunohistochemistry, Flow Cytometry, Derivative Assay

Journal: Scientific reports

Article Title: MHCII reduction is insufficient to protect mice from alpha-synuclein-induced degeneration and the Parkinson's HLA locus exhibits epigenetic regulation.

doi: 10.1038/s41598-025-95679-3

Figure Lengend Snippet: Fig. 1. Experimental design and outcomes. Experiments were split between 2 cohorts of LysMCre + I-Abfl/fl (CRE+) or LysMCre-I-Abfl/fl (CRE−) mice. Both cohorts were given stereotaxic unilateral injections of rAAV2/9-human WT α-synuclein (rAAV9 hWT-ASYN) into the substantia nigra and euthanized 4 months later. Peripheral blood mononuclear cells (PBMCs) and deep cervical lymph nodes (DCLN) were collected for flow cytometry, striatum was microdissected and evaluated with western blots and qPCR for immune markers, and caudal brain was post-fixed and evaluated using immunohistochemistry to verify AAV targeting. Cohort 2 had brains collected for flow cytometry analysis. Figure created with BioRender.com.

Article Snippet: RNA extraction and cDNA synthesis and RT-PCR of human PBMCs RNeasy mini kit (Qiagen) was used according to manufacturer’s instructions.

Techniques: Flow Cytometry, Western Blot, Immunohistochemistry

Journal: Scientific reports

Article Title: MHCII reduction is insufficient to protect mice from alpha-synuclein-induced degeneration and the Parkinson's HLA locus exhibits epigenetic regulation.

doi: 10.1038/s41598-025-95679-3

Figure Lengend Snippet: Fig. 3. Reduction of myeloid MHCII in CRE + mice has a global effect on CD4 T-cell subsets. PBMCs were isolated from LysMCre-I-Abfl/fl (CRE−) and LysMCre + I-Abfl/fl (CRE+) mice and evaluated for T cell subsets using flow cytometry. (A) Representative dot blots and quantifications of CD4 + T cell subsets show that naïve CD4 + T cells (CD44-CD62L+) were increased in CRE + animals compared to CRE-, while CD4 + central memory (CD44 + CD62L+) and CD4 + effector memory (CD44 + CD62L-) were decreased in CRE+. (B) Representative dot blots and quantifications of CD8 + T cell subsets show there was only a trend to increase (p = 0.0873) in CD8 + naïve T cells, with no difference in CD8 + central memory and effector memory cells. (C,D) Yet, frequencies of CD4+ and CD8+ T cells within the CD3 + population were not different between genotypes. (E) Representative dot blots and quantification display a decrease in Tregs (Tbet-FoxP3+) in CRE + animals. Data are plotted as mean ± standard error of the mean (SEM). P values are indicated for unpaired two-tailed t-test (Welch’s correction was used for those measures with significantly different variances): *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: RNA extraction and cDNA synthesis and RT-PCR of human PBMCs RNeasy mini kit (Qiagen) was used according to manufacturer’s instructions.

Techniques: Isolation, Flow Cytometry, Two Tailed Test

Journal: Scientific reports

Article Title: MHCII reduction is insufficient to protect mice from alpha-synuclein-induced degeneration and the Parkinson's HLA locus exhibits epigenetic regulation.

doi: 10.1038/s41598-025-95679-3

Figure Lengend Snippet: Fig. 7. The high-risk HLA-DRA rs31298882 GG genotype is associated with increased inducibility of MHCII proteins in cryorecovered PBMCs. Individuals with PD with the high-risk rs3129882GG genotype show greater IFN-γ inducibility compared to AA individuals. HLA-DR and -DQ alpha and beta chain gene expression was measured from cryopreserved PBMCs following thawing and stimulation with vehicle and IFN-γ. Gene expression fold change was quantified using the double delta CT method relative to GAPDH and control AA group with RT-PCR. Inducibility from IFN-γ when compared to vehicle treatment was found in (A) HLA- DRA, (B) HLA-DRB, (D) HLA-DQB in control AA (CAA) and Parkinson’s disease GG (PDGG), (C) yet this effect was only seen in CAA samples when evaluating HLA-DQA. (B) HLA-DRB inducibility was significantly increased in PDGG compared to PDAA individuals. A two-way ANOVA with uncorrected Fisher’s LSD multiple comparisons was used to evaluate HLA expression between groups (control/PD with either AA or GG) and cell treatment (vehicle vs. IFN-γ). *p < 0.05, **p < 0.01.

Article Snippet: RNA extraction and cDNA synthesis and RT-PCR of human PBMCs RNeasy mini kit (Qiagen) was used according to manufacturer’s instructions.

Techniques: Gene Expression, Control, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Scientific reports

Article Title: MHCII reduction is insufficient to protect mice from alpha-synuclein-induced degeneration and the Parkinson's HLA locus exhibits epigenetic regulation.

doi: 10.1038/s41598-025-95679-3

Figure Lengend Snippet: Fig. 8. The high-risk rs3129882GG genotype is associated with increased frequency of CD4 + T cells independent of disease while the low-risk AA genotype is associated with decreased naïve CD8+ and increased CD8+ effector T cells, but only in individuals with PD. Flow cytometry staining was used to determine frequencies of T cells in cryopreserved PBMC samples from healthy controls (HC) and individuals with PD with the rs3129882 genotype AA or GG (increased risk of PD). HC AA n = 19, HC GG n = 6, PD AA n = 25, PD GG n = 23. (A–C) No effect of disease or genotype was identified in frequency of CD3 + T cells, T regulator cells (CD3 + CD4 + CD25 + CCR6+), or TH17 cells (CD3 + CD4 + CD25 + CCR6 + CCR4+) out of total live cells. (D) Ratio of CD4:CD8 cell frequency demonstrates no differences between groups. (E,F) An effect of SNP genotype was seen in the frequency of total CD8 + T cells out of CD3 + cells (p = 0.0514) and CD8 + central memory (CD8 + CCR7 + CD45RA−) out of CD8 + cells (p = 0.0451). (G–I) CD8 + cell subtype frequencies were evaluated and CD8 + naïve (CD8 + CCR7 + CD45RA+) and effector cells (CD8 + CCR7-CD45RA+) differed between disease groups when expressing the AA genotype, while no effect was found in effector memory cells (CD8 + CCR7-CD45RA−). (J) Frequency of CD4 + cells out of CD3 + cells is significantly higher in those with the GG genotype compared to PD AA. (K,L) Evaluation of CD4 + cell subtype frequencies found no differences in central memory (CD4 + CCR7 + CD45RA−), naïve (CD4 + CCR7 + CD45RA+) or effector memory (CD4 + CCR7-CD45RA−) T cells, but increase effector cells (CD4 + CCR7-CD45RA+) in PD AA compared to HC AA. Two-way ordinary ANOVA with Tukey correction for multiple comparisons was used to test for significant differences. Data are plotted as mean ± standard error of the mean. Two-way ordinary ANOVA with Tukey correction for multiple comparisons used to analyze the data. Trending or significant disease, SNP, and interaction effects noted below each graph. *p < 0.05.

Article Snippet: RNA extraction and cDNA synthesis and RT-PCR of human PBMCs RNeasy mini kit (Qiagen) was used according to manufacturer’s instructions.

Techniques: Flow Cytometry, Staining, Expressing