Journal: Blood

Article Title: Patients with CD3G mutations reveal a role for human CD3γ in T reg diversity and suppressive function

doi: 10.1182/blood-2018-02-835561

Figure Lengend Snippet: T-cell activation and cell division following mitogen stimulation. (A) Representative plots and (B) cumulative percentage of (top) CD25 expression, (middle) Ki67 expression, and (bottom) CFSE staining in CD4+ T cells from controls (solid gray) or CD3G patients (red) in unstimulated samples or following activation with PHA, anti-CD3, or anti-CD3/CD28. (C) Representative plots and (D) cumulative data for (top) CD25 expression, (middle) Ki67 expression, and (bottom) CFSE staining in CD8+ T cells from controls (solid gray) or patients (red) among the indicated groups. (A-D) Response to anti-CD3+anti-CD28 was studied in 5 patients (P1-P5) because not enough cells were available from P6. Dose titration curve of CFSE dilution among (E) CD4+ and (F) CD8+ T cells following stimulation with PHA (top) or anti-CD3 (bottom) for patient P6 (red line) and control sample (blue line). Unstim, unstimulated.

Article Snippet: PBMC were isolated to form a single cell suspension and then stained with carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher, Carlsbad, CA) at a concentration of 5 μM/mL for 10 minutes at 37°C in phosphate-buffered saline.

Techniques: Activation Assay, Expressing, Staining, Titration, Control

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 3. Fn facilitates anti-PD-1 efficacy by systematically activating CD8+ TILs in germ-free immunohumanized mice bearing subcutane- ous MSS CRC xenografts and SPF mice bearing orthotopic MSS CRC allografts (A) Model design for germ-free, CD34+-humanized mice bearing subcutaneous HT29 xenografts and experimental groups (n = 6/group). Mice received Fn, Streptococcus mutans UA159 (UA159), or PBS by gavage 3 times a week. Related results can be found in B–F. (B) Engraftment of human immune cells in CD34+-humanized mice were evaluated 14 weeks post implantation (n = 6/group). (C) Representative tumor morphology, weight, and volume (n = 6/group). Red circles indicate the location of subcutaneous tumors. (D) Ki-67, PCNA, and TUNEL staining of HT29 xenografts (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti-PCNA, or TUNEL kit. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. (E) CD8 and PD-1 co-immunofluorescence staining (n = 4/group) and PD-1 IHC in HT29 xenografts (n = 6/group). Tumor tissues were stained using DAPI, Alexa Fluor 647 anti-human CD8, anti-mouse PD-1, and Alexa Fluor 488 anti-Rabbit IgG H&L antibodies for immunofluorescence staining, and anti-PD-1 for IHC. For immunofluorescence staining, red: CD8+ cells, green: PD-1+ cells, blue: DAPI+ cells, yellow: PD-1+ CD8+ cells. For PD-1 IHC staining, blue: hematoxylin+ cells, brown: PD-1+ cells. (F) Flow cytometry analyses of infiltration of CD8+ T cells in HT29 xenografts, and PD-1, IFN-g, TNF-a, and GZMB expression in CD8+ TILs (n = 6/group). (G) Model design for SPF BALB/c mice bearing orthotopic CT26 allografts and experimental groups. Mice were gavaged with Fn conditioned medium (CM), UA159 CM (UACM), or broth Ctrl daily, with anti-PD-1 mAb (aPD-1) or IgG twice a week (n = 8–12/group). Red circles indicate the location of orthotopic tumors. Related results can be found in (H) and (I). (H) Representative tumor morphology, weight, and volume of CT26 allografts (n = 8–12/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: TUNEL Assay, Staining, Immunohistochemistry, Flow Cytometry, Expressing

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 4. Fn decreases PD-1 expression in CD8+ T cell through its small-molecule metabolites (A) Effects of Fn CM fractions on decreasing PD-1 expression in CD8+ T cell after co-culture for 18 h, as determined by flow cytometry (n = 6/group). Fn CM PK: Fn CM treated with proteinase K; >100 kDa: Fn CM > 100 kDa fraction; 10–100 kDa: Fn CM 10–100 kDa fraction; 3–10 kDa: Fn CM 3–10 kDa fraction; <3 kDa: Fn CM < 3 kDa fraction. (B) Workflow to define candidate metabolites from Fn culture medium that suppress PD-1 expression in CD8+ T and promote CD8+ T cell activation in vitro. (C) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot based on non-targeted metabolomic analysis of Fn CM < 3 kDa vs. control (left) and Fn gavage vs. PBS gavage germ-free mouse feces (right) (n = 6/group). (D) Venn diagram of enriched metabolites in Fn CM < 3 kDa vs. control CM, and Fn gavage vs. PBS gavage in germ-free mouse feces, and metabolomic heatmap of metabolites enriched after Fn gavage vs. PBS gavage germ-free mouse feces as determined by untargeted metabolomics (n = 6/group). (E) Targeted metabolomic analysis of short-chain fatty acids (SFCAs) in Fn- and PBS-gavaged germ-free mouse feces (left). Butyric acid quantification in CM and feces from germ-free mice (right) (n = 6/group). (F) Effect of sodium butyrate (NaB) on the expression of PD-1 and GZMB in splenic CD8+ T cells from tumor-bearing mice (n = 6/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Expressing, Co-Culture Assay, Cytometry, Activation Assay, In Vitro, Control

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 5. Fn boosts response to anti-PD-1 therapy through its metabolite butyric acid (A) Workflow for the construction of FN0271 mutant Fn strain deficient in butyric acid biosynthesis. PCR products of FN0271 were analyzed by agarose gel electrophoresis to confirm the insertion mutant of Fn (left). GC-MS was conducted to detect butyric acid levels in Fn CM and FN0271 mutant (Mut) Fn CM (right). (B) Effect of Fn CM, Mut Fn CM, and NaB on PD-1 and GZMB expression in splenic CD8+ T cells (n = 6/group). (C) Evaluation of T cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) assay and flow cytometry (n = 6/group). (D) Workflow to assess Fn CM and Mut Fn CM on anti-PD-1 mAb efficacy in vitro using CD8+ TILs from CT26 tumor-bearing mice. (E) Effect of Fn CM and Mut Fn CM in combination with anti-PD-1 mAb (aPD-1) on PD-1, GZMB, and IFN-g expression in CD8+ TILs (n = 6/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S6.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Mutagenesis, Agarose Gel Electrophoresis, Gas Chromatography-Mass Spectrometry, Expressing, CFSE Assay, Cytometry, In Vitro

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 6. Fn boosts anti-PD-1 mAb tumor-killing effects through its metabolite butyric acid in autologous co-culture system of PBMCs with tumor organoids from patients with MSS CRC (A) Schematic diagram for the evaluation of Fn CM and metabolites on anti-PD-1 therapy-mediated killing of CRC organoids with autologous peripheral blood mononuclear cell (PBMC)-derived CD8+ T cells. (B) Representative images of anti-PD-1 (aPD-1) therapy combined with Fn CM, FN0271 Mut Fn CM, or NaB on tumor killing efficiency. MSS CRC organoids (red) were labeled with CellTrace Far Red and apoptotic cells were labeled with green caspase-3/7 probe (n = 6/group). Black circle: organoid, small dot: T cell, red: CellTrace far red+ cells, green: caspase-3/7+ cells. (C) Apoptosis in CRC organoids, as determined by flow cytometry (n = 6/group). (D) Flow cytometry of PD-1, GZMB, and IFN-g in autologous CD8+ T cells (n = 3/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S7 and Table S2.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Co-Culture Assay, Derivative Assay, Labeling, Cytometry, Flow Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 7. Fn-derived butyrate facilitates anti-PD-1 therapy in MSS CRC in a CD8+ T cell-dependent manner (A) CT26 subcutaneous allografts tumor-bearing BALB/c mice received NaB in drinking water, together with anti-PD-1 mAb (aPD-1), anti-CD8a mAb, or IgG twice a week (n = 6–10/group). Related results can be found in B–H. (B) Representative tumor gross morphology, tumor weight, and tumor volume. NaB potentiated with anti-PD-1 to suppress tumor growth, an effect abolished by the depletion of CD8+ T cells by anti-CD8a (n = 6–10/group). Red circles indicate the location of subcutaneous tumors. (C) Tumoral butyrate among different groups (n = 6/group). (D) TUNEL, (E) Ki-67, and PCNA IHC in CT26 allografts to analyze cell apoptosis and proliferation (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti- PCNA, or TUNEL kit. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. (F) Infiltration of CD8+ TILs in CT26 allografts, as determined by flow cytometry and (G) IHC (n = 6/group). Tumor tissues were stained using anti-CD8. Blue: hematoxylin+ cells, brown: CD8+ cells.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 8. Fn metabolite butyric acid regulates CD8+ T cell through an epigenetic mechanism leading to upregulation of TBX21 (T-bet) (A) CD8+ T cells treated with Fn CM, FN0271 Mut Fn CM, and control CM were analyzed by RNA sequencing. Principal component analysis (PCA) and heatmap analysis of differentially expressed genes, and validation by qPCR (n = 3/group). qPCR data were normalized using Actb as an internal control.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Control, RNA Sequencing, Biomarker Discovery

Journal: Clinical and Experimental Immunology

Article Title: Interleukin‐2 reverses CD8 + T cell exhaustion in clinical malignant pleural effusion of lung cancer

doi: 10.1111/cei.12845

Figure Lengend Snippet: CD8+ T cells in the MPE of LC show the lowest proliferation capacity. (a) Proliferation of CD3+CD8+ T cells [stained with carboxy fluorescein succinimidyl ester (CFSE)] in PB of healthy control and LC patients, as well as in the MPE of LC patients. (b) The percentage in each generation with CFSE dilution. DT = division time; MFI = mean fluorescent intensity; DI% = division percentage; PI = proliferation index; DI = division index. The non‐paired t‐test, *P < 0·05, PB versus MPE.

Article Snippet: The MC in the MPE and PBMC were stained with carboxy fluorescein succinimidyl ester (CFSE; Dojindo Laboratories, Shanghai, China); 1 × 10 7 PBMC/MC were incubated in phosphate‐buffered saline (PBS) with a final concentration of CFSE (2 μM) for 20 min at 37°C.

Techniques: Staining, Control

Journal: Frontiers in Immunology

Article Title: Increased CD14 + HLA-DR −/low Myeloid-Derived Suppressor Cells Correlate With Disease Severity in Systemic Lupus Erythematosus Patients in an iNOS-Dependent Manner

doi: 10.3389/fimmu.2019.01202

Figure Lengend Snippet: The expression of M-MDSCs in SLE patients. (A) Representative flow cytometric speckle maps showed the expression of M-MDSCs in newly diagnosed SLE patients and healthy controls. (B) M-MDSCs levels in newly diagnosed SLE patients and healthy controls (in CD14 + monocytes). (C) M-MDSCs levels in newly diagnosed SLE patients and healthy controls (in PBMCs). (D) MDSCs had a significantly immunosuppressive activity on CD4 + T cells as compared to the CD14 + HLA-DR + cells from active SLE patients. The proliferation of CD4 + T cells was measured by dilution of CFSE staining intensity using flow cytometry. (E) IFN-γ-producing was also significantly decreased in CD4 + T cells co-cultured with M-MDSCs. Each point represents an individual. The horizontal bar represents the average. ** P < 0.01.

Article Snippet: CD4 + T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) (0.5 μmol/L) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Activity Assay, Staining, Flow Cytometry, Cell Culture

Journal: Frontiers in Immunology

Article Title: Increased CD14 + HLA-DR −/low Myeloid-Derived Suppressor Cells Correlate With Disease Severity in Systemic Lupus Erythematosus Patients in an iNOS-Dependent Manner

doi: 10.3389/fimmu.2019.01202

Figure Lengend Snippet: Effects of M-MDSCs on the proliferation of autologous T cells and production of IFN-γ. (A) Plasma-induced MDSCs had a significant immunosuppressive activity on CD4 + T cells in a dose-dependent manner as compared to the CD14 + HLA-DR + cells. The proliferation of CD4 + T cells was measured by dilution of CFSE staining intensity using flow cytometry. (B) IFN-γ-production was also significantly decreased in CD4 + T cells co-cultured with M-MDSCs. (C) Representative flow cytometry flow histograms demonstrated the immunosuppressive activity of M-MDSCs and CD14 + HLA-DR + cells. (D,E) Effects of iNOS and Arg-1 inhibitors on the M-MDSC suppression with respect to CD4 + T cell proliferation and IFN-γ production. Ctrl, control ; * P < 0.05; ** P < 0.01.

Article Snippet: CD4 + T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) (0.5 μmol/L) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).

Techniques: Clinical Proteomics, Activity Assay, Staining, Flow Cytometry, Cell Culture, Control

Journal: European Journal of Histochemistry : EJH

Article Title: Galunisertib enhances chimeric antigen receptor-modified T cell function

doi: 10.4081/ejh.2020.3122

Figure Lengend Snippet: Galunisertib did not affect T cell proliferation. (A) and (B) NT cells, (C) CD133-CAR T cells, and (D) HER2-CAR T cells were labeled with 0.5 μM CFSE. (A) NT cells were cultured with (+Transact) or without Transact (control). B-D) Different T cells were activated with Transact, irradiated U251 CD133-OE luc and MDA-MB-453 luc cells, respectively. Meanwhile, Galunisertib was added, and DMSO or culture medium (R-10) was used as control. After four days, CFSE signals in T cells were detected by flow cytometry.

Article Snippet: CD133-CAR T cells, HER2-CAR T cells, and NT cells were labeled with 0.5 μM CFSE (Carboxyfluorescein Diacetate Succinimidyl Ester, 65-0850-84, ThermoFisher Scientific) according to the method provided by the CFSE manufacturer.

Techniques: Labeling, Cell Culture, Control, Irradiation, Flow Cytometry

Journal: Scientific reports

Article Title: Establishment of a novel human lymphoblastic cell strain with the long arm of chromosome 11 aberration without MLL rearrangement.

doi: 10.1038/s41598-017-00874-6

Figure Lengend Snippet: Figure 6. Cytokines secreted by CHH-1 cells and their proliferative effect on the cells. (A) The concentration of different cytokines in the culture supernatant secreted by CHH-1 cells as compared with MC116, Raji and Toledo cells as detected by ELISA. (B) Transcription of the secreted cytokines and their corresponding receptors is detected in CHH-1 cells by RT-PCR. GAPDH is used as an internal control. (C) The effect of the cytokines on CHH-1 cell proliferation is determined by CFDA-SE assay. Values represent the mean ± SD of three independent experiments.

Article Snippet: The proliferative response of CHH-1 cells to cytokines rhIL-2 (50 ng/ml), rhIL-10 (100 ng/ml), rhIL-22 (100 ng/ml), rhLIF (100 ng/ml), rhTNF-α(50 ng/ml) and rhFlt3lig (100 ng/ml) was examined by carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) Cell Proliferation Assay (Beyotime).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: A lncRNA landscape in breast cancer reveals a potential role for AC009283.1 in proliferation and apoptosis in HER2-enriched subtype

doi: 10.1038/s41598-020-69905-z

Figure Lengend Snippet: ( A ) Real-time qPCR illustrating gene silencing by shRNA against AC009283.1 in SKBR3, a HER2-enriched cell line model. ( B ) Supervised hierarchical cluster analysis shows 158 differentially expressed genes after AC009283.1 knockdown (Foldchange < − 1.2, > 1.2 p-value < 0.05); we observed 94 up-regulated and 64 down-regulated genes in shRNA-2 vs NC and ( C ). Their distribution across the genome. ( D ) Ingenuity Pathway Analysis of 158 genes differentially expressed after knockdown of AC009283.1 in SKBR3 cells. ( E ) Enrichment pathway analysis of genes differently expressed in samples of HER2-enriched tumors from TCGA with high vs low expression of AC009283.1.

Article Snippet: Human BRCA cell lines included in the analyses (MCF7, ZR-75-1, MDA-MB-361, SKBR3, MDA-MB-468, HCC1187, HS578T, MDA-MB-231, MDA-MB-453) and non-tumorigenic epithelial cell line MCF10A, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: shRNA, Knockdown, Expressing

Journal: Scientific Reports

Article Title: A lncRNA landscape in breast cancer reveals a potential role for AC009283.1 in proliferation and apoptosis in HER2-enriched subtype

doi: 10.1038/s41598-020-69905-z

Figure Lengend Snippet: ( A ) Proliferation assay performed following knockdown of AC009283.1 with shRNA2, shRNA NC as negative control, and wildtype cell line. Cells were counted every day for four days. *p < 0.05, **p < 0.005, ***p < 0.0005, comparing with shRNA NC for each condition via Student’s t test. ( B ) Proliferation index using CFSE assay in SKBR3 cells with shRNA2 AC009283.1 and shRNA NC. *p < 0.05, **p < 0.005, ***p < 0.0005, comparing with shRNA NC for each condition via Student’s t test. ( C ) Representative CFSE flow-cytometry histograms showing the effect on in vitro SKBR-3 cell proliferation with shRNA NC and shRNA2 (AC009283.1 knock-down). ( D ) Cell cycle assay with flow-cytometry using (propidium iodide) PI on in vitro SKBR-3 cells, AC009283.1 knockdown causes accumulation of cells at S phase. *p < 0.05, **p < 0.005, ***p < 0.0005, comparing to shRNA NC for each condition via Student’s t test.

Article Snippet: Human BRCA cell lines included in the analyses (MCF7, ZR-75-1, MDA-MB-361, SKBR3, MDA-MB-468, HCC1187, HS578T, MDA-MB-231, MDA-MB-453) and non-tumorigenic epithelial cell line MCF10A, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Proliferation Assay, Knockdown, shRNA, Negative Control, CFSE Assay, Flow Cytometry, In Vitro, Cell Cycle Assay

Journal: PLoS ONE

Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

doi: 10.1371/journal.pone.0074363

Figure Lengend Snippet: A. sCAR-PPAb did not enhance the phagocytosis of K562 cells by macrophages. K562 cells stained with CFSE and pretreated with sCAR-PPAb or PBS were injected into the abdominal cavity in ICR mice. After 5h, cells in the abdominal cavity were then collected and stained with a primary antibody against F4/80 or IgG isotype followed by a goat anti-rat IgG-PE antibody. Cells were then analyzed by flow cytometry. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p>0.05, n=3). B. sCAR-PPAb enhanced the phagocytosis of K562/ADR by macrophages. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p<0.05, n=3). C. The cytotoxicity of sCAR-PPAb on K562 and K562/ADR was analyzed by MTT assay. D. Mouse macrophages from the abdominal cavity were isolated and cultured in 96-well plates. The cells were then treated with 5μg of sCAR-PPAb for 5 hours. PBS treatment served as the control. The cell viability was analyzed by MTT assay.

Article Snippet: K562/ADR cells were stained with carboxyl fluorescein succinimidyl ester (CFSE), and 1 x 10 6 cells were mixed with 10μg of a mouse anti-SLMAP mAb (Santa Cruz Biotechnology) or a mouse IgG (Sigma-Aldrich, Co., St. Louis, MO, USA) followed by adding into wells pre-cultured with macrophages.

Techniques: Staining, Injection, Flow Cytometry, MTT Assay, Isolation, Cell Culture, Control

Journal: PLoS ONE

Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

doi: 10.1371/journal.pone.0074363

Figure Lengend Snippet: A. sCAR-PPAb combined with a CAR antibody was used in immunoprecipitation. PBS combined with CAR antibody served as the control. Precipitates were analyzed by Western blot for SLMAP. Whole cell lysis (WCL) was monitored for Actin levels. B. SLMAP levels on K562 and K562/ADR were determined by Western blot. Actin served as a loading control. C. A SLMAP antibody enhanced the phagocytosis of K562/ADR by macrophages in vitro. An IgG isotype served as the control. The portion of macrophages ingesting CFSE stained K562/ADR cells was observed under a fluorescence microscope (200 x). A significant enhancement of phagocytosis upon SLMAP antibody treatment was determined (p<0.05).

Article Snippet: K562/ADR cells were stained with carboxyl fluorescein succinimidyl ester (CFSE), and 1 x 10 6 cells were mixed with 10μg of a mouse anti-SLMAP mAb (Santa Cruz Biotechnology) or a mouse IgG (Sigma-Aldrich, Co., St. Louis, MO, USA) followed by adding into wells pre-cultured with macrophages.

Techniques: Immunoprecipitation, Control, Western Blot, Lysis, In Vitro, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection

doi: 10.1101/2021.07.08.451596

Figure Lengend Snippet: Effects of the recombinant protein NMHC on CD8 + T cell responses. A, to verify that NMH antigens can be effectively internalized into splenocytes, splenocytes prepared from naïve BALB/c mice were co-cultured with Alexa Fluor 488-labeled NMH or NMH alone and detected by fluorescent inverted microscope. B, then, fresh isolated naïve splenocytes were divided into two parts. One part was pulsed with 5 μg/mL NMH, labeled with 10 μM of CFSE and termed as CFSE high target cells, the other part was loaded with BSA, labeled with 1 μM of CFSE and defined as CFSE low control cells, both of them detected by fluorescent inverted microscope and flow cytometry. C, the equal number of two parts cells were mixed and injected into recipient mice. Twelve hours later, the splenocytes from these mice were used to examine the antigen specific cytolytic responses. D, the mRNA levels of perforin and granzyme of recipient mice were examined by qPCR. Statistical analyses were performed using Student’s t-test and one-way ANOVA. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ns, not significant.

Article Snippet: CFSE Cell Tracking Kit was purchased from Beyotime Biotechnology (Haimen, Jiangsu, China) BDTM.

Techniques: Recombinant, Cell Culture, Labeling, Inverted Microscopy, Isolation, Control, Flow Cytometry, Injection