Journal: Current Protocols

Article Title: Density Gradient Centrifugation‐Independent Purification of Human Basophils

doi: 10.1002/cpz1.991

Figure Lengend Snippet: Yield, purity, and viability of isolated human basophils. ( A ) Basophils yield was calculated by extrapolating the counted number of FcεRIα and CD123 double‐positive cells in the isolated cell fraction to the volume of blood from which the basophils were isolated (n = 34). ( B ) Purity of basophils was determined by measuring the percentage of FcεRIα and CD123 double‐positive cells through flow cytometry (n = 41). ( C ) Viability of basophils directly after isolation was determined through the exclusion of 7AAD‐positive cells (n = 8). ( D ) Representative dot plot demonstrating typical viability of basophils directly after isolation was determined through measuring the percentage of 7AAD‐positive cells. ( E‐G ) Gating strategy to identify basophils through flow cytometry analysis in a representative sample. ( E ) Side scatter (SSC‐A) and forward scatter (FSC‐A) properties for detecting basophil‐sized cells after isolating. ( F ) Exclusion of duplets. ( G ) Identification of basophils through measuring FcεRIα and CD123 double‐positive cells.

Article Snippet: Purified human basophil suspension (see ) PBS w/o Ca 2+ and Mg 2+ (ROTH, cat. no. 9143.2) Pacific Blue (PB)‐conjugated anti‐human CD123 antibody (Biolegend, cat. no. 306044, RRID: AB_2750165) Allophcocyanin (APC)‐conjugated anti‐human FcεRIα antibody (Biolegend, cat. no. 334612, RRID: AB_10578086) 7‐AAD staining solution (Miltenyi, cat. no. 130‐11‐568) 1.5‐ml microcentrifuge tubes (Sarstedt, cat. no. 72.690.001) Flow cytometer (Beckman Coulter, Cytoflex S)

Techniques: Isolation, Flow Cytometry

Journal: Aging Cell

Article Title: Legumain deficiency halts atherogenesis by modulating T cell receptor signaling

doi: 10.1111/acel.14391

Figure Lengend Snippet: Deletion of legumain reduces survival and increases apoptosis of CD4+ T cells (a) Quantification of co‐stimulatory molecule expression in CD4 + T cells from each group. The data were obtained by quantitative real‐time PCR ( n = 7 mice per group). (b) The percentages of CD44 − CD62L + naive (NA), CD44 + CD62L − effector memory (EM), and CD44 + CD62L + central memory (CM) T cells in gated splenic CD4 + T cells from Lgmn +/+ Apoe −/− mice and Lgmn −/− Apoe −/− mice were quantified ( n = 5 mice per group). (c–e) Representative flow cytometry histograms for Ki‐67. The expression of Ki‐67 in CD4 + T cells in each group was assessed by quantitative real‐time PCR and flow cytometry analysis ( n = 7 mice per group). (f, g) Representative flow cytometry gating strategies for Annexin V staining. The percentage of live CD4 + T cells in each group was assessed directly at harvest ( n = 5 mice per group). (h, i) TdT‐mediated dUTP nick‐end labeling (TUNEL) apoptosis assay. Double‐labeling immunofluorescence of TUNEL (green) and CD4 (red) in spleen sections from Lgmn +/+ Apoe −/− mice and Lgmn −/− Apoe −/− mice. The number of TUNEL + CD4 + T cells in each field was quantified ( n = 5 mice per group). CM, central memory; EM, effector memory; NA, naive T cells; TUNEL, TdT‐mediated dUTP nick‐end labeling. Data information: The exact p value is specified. The p value was determined by an unpaired two‐tailed Student's t test (a, b, d, e, g, and i).

Article Snippet: Apoptosis staining was performed via an Annexin V Apoptosis Detection Kit (Elabscience, E‐CK‐A212) according to the manufacturer's instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Staining, End Labeling, TUNEL Assay, Apoptosis Assay, Labeling, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Stress-Survival Pathway Profiling Reveals MCM10 as a Candidate Biomarker of Hispanic Colorectal Cancer Disparities

doi: 10.64898/2026.01.06.698057

Figure Lengend Snippet: ( A-B). The flow cytometric analysis was performed to evaluate the cell cycle arrest after MCM10 knockdown in both Caco-2 and SW480 cells. The depletion of MCM10 significantly reduced the number of cells at G1/G0 and increased at G2/M phase respectively. (C-D). Likewise, the apoptosis level of both cell lines was determined by flow cytometric analysis using annexin V-FITC. The result demonstrated that MCM10 knockdown significantly increased the early and late apoptosis in Caco-2 and SW480 cell lines compared to their respective NC group. ( E-F). The ROS was evaluated to check whether cell cycle arrest and apoptosis process was induced by reactive oxygen or not. The knockdown of MCM10 in Caco-2 and SW480 cells potentially activate the cellular ROS production to promote cell cycle arrest and apoptosis pathway to reduce the carcinogenesis compared to their respective NC group. The results are representative of at least three independent experiments. ( * P<0.05, ** P< 0.01, ***P<0.001 ). Scale bar= 100µm.

Article Snippet: Likewise, the apoptosis was detected using Annexin V Apoptosis Detection Kit with 7-AAD (Tonbo Biosciences, CA, USA).

Techniques: Knockdown

Journal: bioRxiv

Article Title: Stress-Survival Pathway Profiling Reveals MCM10 as a Candidate Biomarker of Hispanic Colorectal Cancer Disparities

doi: 10.64898/2026.01.06.698057

Figure Lengend Snippet: Transcriptomic/proteomic analysis post MCM10 Knockdown in CRC cells and validation of findings in Hispanic organoid. (A-B) . The downstream signaling was evaluated by performing high-throughput RS and high-resolution proteomic- analysis, respectively. The heatmap from RS showed differentially expressed genes from Caco-2 and SW480 cells post-MCM10 knockdown. Overall, 26 and 44 DEGs were identified in Caco-2 and SW480 cell lines with MCM10 knockdown, with a log2 fold change cutoff of >0.95 and p-adjusted value (with FDR correction) of < 0.05. (C). Protein expression of MCM10 after transfection which was evaluated by western blotting where it shows clear downregulation of its expression in MCM10-KD group. (D). The qRT-PCR was performed to evaluate the expression of PPFIA1 as key differentially expressed genes and protein in response to MCM10 KD where it significantly downregulated along with MCM10 in transfected group compared to the NC group. A significant downregulation of PPFIA1 was observed along with MCM10 after the transfection (E-F). The MCM10 KO efficiency was measured by qRT-PCR in transcript level and western blotting in protein level. (G-I) Furthermore, the size of organoid was evaluated after MCM10 KO. The MCM10 KO significantly reduced the organoid size compared to the WT group. Similarly, the live (green fluorescence by premixed acridine orange dye) and dead (red fluorescence by propidium iodide dye) assay revealed that MCM10 KO significantly induces the apoptosis in order to reduce the carcinogenesis. (J) Notably, MCM10 KO significantly reduced the expression of PPFIA1 in Hispanic organoid model which are in line with our cell lines demonstration. The data was shown as meanLJ±LJSEM compared to their negative control group and representative of at least three independent experiments. ( * P<0.05, ** P< 0.01, ***P<0.001 ). Scale bar= 100µm.

Article Snippet: Likewise, the apoptosis was detected using Annexin V Apoptosis Detection Kit with 7-AAD (Tonbo Biosciences, CA, USA).

Techniques: Knockdown, Biomarker Discovery, High Throughput Screening Assay, Expressing, Transfection, Western Blot, Quantitative RT-PCR, Fluorescence, Negative Control