indirect cd133 microbeads  (Miltenyi Biotec)

Journal: STAR Protocols

Article Title: Protocol to generate human stem cell-derived CD70-directed allogeneic CAR-NKT cells for treating renal cell carcinoma

doi: 10.1016/j.xpro.2025.104340

Figure Lengend Snippet: Generation of Allo CAR70-NKT cells from HSPCs using a clinically guided culture method (A and B) Schematics showing the generation of Allo CAR70-NKT cells (A) and the design of Lenti/iNKT-CAR70-IL-15 lentivector (B). (C) FACS detection of NKT TCR expression in lentivector-transduced CD34 + HSPCs. (D) Quantification of (C) (n = 5; n indicates different cord blood donors). (E) FACS monitoring of the generation of Allo CAR70-NKT cells during the 6-week culture. (F) Percentage of Allo CAR70-NKT cells in total live cells during the 6-week culture (n = 5; n indicates different cord blood donors). (G) Yield of Allo CAR70-NKT cells (n = 5; n indicates different cord blood donors). (H) CAR70 expression on Allo CAR70-NKT cells (n = 5; n indicates different cord blood donors). (I) ELISA measurements of IL-15 production by Allo CAR70-NKT cells with or without αGC stimulation (n = 4). (J) CD4/CD8 subpopulations of Allo CAR70-NKT cells. Data generated from 5 cord blood donors are shown. SP, single-positive; DP, double-positive; DN, double-negative. (K–M) Antigen responses of Allo CAR70-NKT cells. Allo CAR70-NKT cells were stimulated with/without αGC-loaded PBMCs for 1 week. (K) Experimental design. (L) Growth curve of Allo CAR70-NKT cells (n = 4). (M) ELISA measurements of effector cytokine levels in the culture supernatants collected on day 5 (n = 4). Representative of over 5 experiments. Data are presented as the mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Student’s t test (I and M).

Article Snippet: Human CD34 MicroBeads Kit , Miltenyi Biotec , CAT#130-046-703.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Generated

Journal: Alzheimer's & Dementia

Article Title: Female‐biased astrocytic priming shapes early locus coeruleus vulnerability in an Aβ oligomer milieu

doi: 10.1002/alz.71168

Figure Lengend Snippet: Astrocyte‐linked C3 elevation and lactate–MCT2–OXPHOS axis engagement in the pons of female APP/PS1 mice. Astrocyte‐enriched pons fraction shows increased C3 expression in 2‐ to 3‐month‐old female APP/PS1 mice. A, Schematic illustrating MACS‐based isolation of pontine astrocytes. Schematic created using Biorender. B, Assessment of C3 expression by qPCR followed by 1% agarose gel electrophoresis–gel image showing C3 bands at 172 bp in astrocyte‐enriched fractions (lane 1–4) and astrocyte‐depleted fractions (lane 5–8). C, D, Bar graphs demonstrating elevated C3 levels in the astrocyte‐enriched fraction of female APP/PS1 mice compared to other groups and to their corresponding negative fraction. Each bar represents pooled pons samples from 8 mice per group. Lactate–MCT2 axis and OXPHOS pathway engagement in female APP/PS1 mice. Female APP/PS1 mice showed higher (E) lactate levels in the pons measured by colorimetric assay and increased (F) MCT2 expression assessed by qPCR ( n = 6 per group; two‐way ANOVA with Tukey post hoc test; data shown as mean ± SEM; * p < 0.05, ** p < 0.01). G, H, mRNA expression profiling of mitochondrial markers by qPCR in the pons. Female APP/PS1 mice showed significantly elevated expression of the mitochondrial complex I subunit Ndufs8 and complex V subunit Atp5a1 , indicating adaptive mitochondrial activation in the pons ( n = 5‐6; two‐way ANOVA with Tukey post hoc test; data shown as mean ± SEM; * p < 0.05, ** p < 0.01). I, Graphical summary. Elevated Aβ oligomers in female APP/PS1 mice shift astrocytes from a resting to an active GFAP +ve state, with associated C3 and NF‐κB2 upregulation and engagement of lactate–OXPHOS axis, supporting increased energetic demands. Graphical summary created using Biorender. Aβ, amyloid beta; ACSA‐1, astrocyte cell surface antigen 1; ANOVA, analysis of variance; Atp5a1, ATP synthase F1 subunit α; C3, complement component 3; GLUT1, glucose transporter 1; MACS, magnetic‐activated cell sorting; MCT2, monocarboxylate transporter 2; Ndufs8, NADH dehydrogenase iron‐sulfur protein 8; NF‐κB2, nuclear factor‐kappa‐B p100/p52 subunit; OXPHOS, oxidative phosphorylation; qPCR, quantitative polymerase chain reaction; SEM, standard error of mean; WT, wild type.

Article Snippet: Cells were incubated with FcR blocking reagent (Miltenyi Biotec, 130‐092‐575) at a 1:9 dilution for 10 minutes to prevent non‐specific antibody binding, followed by incubation with Anti‐GLAST or Astrocyte Cell Surface Antigen‐1 (ACSA‐1)‐Biotin (Miltenyi Biotec, 130‐095‐826) for 10 minutes.

Techniques: Expressing, Isolation, Agarose Gel Electrophoresis, Colorimetric Assay, Activation Assay, FACS, Phospho-proteomics, Real-time Polymerase Chain Reaction