astrocytes cell surface antibody 2 microbead acsa2  (Miltenyi Biotec)

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: Preparation of the D-Bmp2@M system and its therapeutic mechanism for osteoporosis fractures. Bmp2 fused with DSS6 were expressed in HEK293T and then encapsulated in porous PLGA microspheres to construct the D-Bmp2@M system. Upon injection into the osteoporotic fracture site, the system gradually releases D-Bmp2 as the microspheres degrade over approximately 30 days. The released D-Bmp2 actively binds to bone tissue due to the affinity of DSS6 for bone. This localized enrichment promotes osteogenic activity at the fracture site, promoting fracture healing while reducing the risk of ectopic bone formation. The sustained-release and targeted delivery systems provides a superior therapeutic strategy for fracture treatment.

Article Snippet: We observed the healing properties of the microsphere surface using a JSM-6700F scanning electron microscope (SEM, JEOL, Japan).

Techniques: Construct, Injection, Activity Assay

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: Preparation and characterization of self-healing sustained-release microspheres loaded with D-Bmp2. a. Representative SEM images of microspheres before (top) and after (bottom) healing; left scale bar: 10 μm; middle and right scale bar: 2.5 μm. b. Statistical analysis of the microsphere diameter before and after healing determined via SEM. c. Representative confocal microscopy images of protein-loaded microspheres: PLGA microspheres (red) and Cy5-labeled D-Bmp2 (blue). Scale bar: 2 μm. d. Morphology of lyophilized D-Bmp2@M powder. e. SDS-PAGE of lyophilized D-Bmp2@M powder at different storage times. f. Representative firefly luciferase images from bioactivity assays of lyophilized D-Bmp2@M powder at different times. g. Activity change curve of lyophilized D-Bmp2@M powder at different time points (n = 3 per group). h, i. In vitro fluorescence intensity changes of Cy7-labeled D-Bmp2 from microspheres: (h) Representative fluorescence images of Cy7-D-Bmp2 maintained in microspheres (0–30 days) (top) and representative SEM images of microsphere degradation at different time points. Scale bar: 2.5 μm (bottom); (i) Relative fluorescence intensity change of Cy7-D-Bmp2 maintained in microspheres (n = 3 per group). j. Representative firefly luciferase images from Bmp2 reporter assays. k. Protein activity normalization: ratio of luminescence intensity (data from ) to protein concentration (data from ) (n = 3 per group). The data are presented as the means ± SDs. One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant).

Article Snippet: We observed the healing properties of the microsphere surface using a JSM-6700F scanning electron microscope (SEM, JEOL, Japan).

Techniques: Confocal Microscopy, Labeling, SDS Page, Luciferase, Activity Assay, In Vitro, Fluorescence, Protein Concentration

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: In vitro validation of D-Bmp2@M osteogenic efficacy and inhibition of ectopic ossification. a. Schematic diagram of the osteoblast-bone Transwell model. Bmp2/D-Bmp2@M microspheres or free Bmp2/D-Bmp2 were loaded in the upper chambers, MC3T3-E1 cells were cultured on two coverslips (one of which was precoated with HA) in the lower compartments, and the medium was refreshed every day for 7 or 14 days. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed at days 7 and 14, respectively. b. Osteogenic differentiation staining: ALP (early-stage, day 7) and ARS (late-stage, day 14) staining. Scale bar: 200 μm. c. ALP activity was quantitatively analyzed using an ALP kit (n = 3 per group). d. Relative quantitative analysis of ARS staining was performed at an OD of 562 nm (n = 3 per group). e. qPCR analysis of Bmp2 signaling-related mRNA in MC3T3-E1 cells (n = 3 per group). f. Schematic diagram of the muscle-bone Transwell model. Bovine bone slices were co-incubated with Bmp2/D-Bmp2@M or free Bmp2/D-Bmp2 in the upper chambers, and C2C12 cells were cultured in the lower chambers and the medium was refreshed every day for 7 days. D-Bmp2 and Bmp2 retention on bone slices and ALP staining of C2C12 cells were analyzed on day 7. g. Representative fluorescence imaging of bone slices incubated with AF647-conjugated anti-Flag antibodies (above) (yellow arrows: bone slice) and C2C12 ALP staining images (below), scale bar: 200 μm. h. AF647-conjugated anti-Flag antibody fluorescence intensity quantification in bone slices (n = 3 per group). i. Quantification of ALP activity in C2C12 cells (n = 3 per group). j. qPCR analysis of Bmp2 signaling-related mRNA in C2C12 cells (n = 3 per group). The data are presented as the means ± SDs. One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: We observed the healing properties of the microsphere surface using a JSM-6700F scanning electron microscope (SEM, JEOL, Japan).

Techniques: In Vitro, Biomarker Discovery, Inhibition, Cell Culture, Staining, Activity Assay, Incubation, Fluorescence, Imaging

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: In vivo testing of release kinetics and bone accumulation of D-Bmp2@M. a. Representative fluorescence images showing the changes in Cy7 fluorescence after local injection. b. Quantitative analysis of the changes in relative fluorescence intensity (n = 6 per group). c. Representative ex vivo fluorescence images of bone tissues at 1 day post-injection of free Cy7-D-Bmp2 or Cy7-Bmp2, along with quantitative analysis of the bone fluorescence intensity (n = 6 per group). d. Representative IFHC images at 1 day post-injection showing Bmp2 or D-Bmp2 localization and the bone to muscle fluorescence intensity ratio (n = 6 per group). IFHC: anti-Flag antibody (yellow), DAPI (blue); the white dotted line represents the boundary between bones and muscles (M: muscle, B: bone); scale bar: 20 μm. e. Representative ex vivo fluorescence images of bone tissues at 6 days post-injection of PLGA microspheres loaded with Cy7-D-Bmp2 or Cy7-Bmp2, along with quantitative analysis of the bone fluorescence intensity (n = 6 per group). f. Representative IFHC images at 6 days post-injection showing Bmp2 or D-Bmp2 localization and the bone-to-muscle fluorescence intensity ratio (the fluorescence intensity of the 10-μm bone boundary to muscle tissue) (n = 6 per group). IFHC: anti-Flag antibody (yellow), DAPI (blue); the white arrows highlight PLGA microspheres; the white dotted line represents the boundary between bones and muscles (M: muscle, B: bone); scale bar: 20 μm. The data are presented as the means ± standard deviations (SDs). Unpaired Student's t -test was used for two-group comparisons. Significance levels: ∗∗∗∗ p < 0.0001.

Article Snippet: We observed the healing properties of the microsphere surface using a JSM-6700F scanning electron microscope (SEM, JEOL, Japan).

Techniques: In Vivo, Fluorescence, Injection, Ex Vivo, Muscles

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

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of differentiation heterogeneity in the OCI-AML8227 cell line model. This cell line produces leukemic blasts expressing immature myeloid markers (CD34) as well as differentiated monocytic markers (CD64 and CD14). CD38 is a transient myeloid differentiation marker, which is initially expressed in early myeloid progenitor cells. b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 96 hours along an 8-point dose curve with venetoclax. Cell viability was assessed by CellTiter Aqueous colorimetric assay. c. AUC values from the dose-response curves for each immunophenotypic population. Significance was evaluated using a two-tailed t-test. d–i. Cell surface expression of CD34, CD38, CD64, and CD14 in OCI-AML8227 cells cultured in triplicate for 72 hours with 1 μM venetoclax or an equivalent volume of DMSO. Cells were analyzed either as immunomagnetically fractionated populations (CD34-enriched and CD34-depleted) or as unfractionated cells. Significance was evaluated using ordinary one-way ANOVA followed by Holm-Šidák post-test correction. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Marker, Cell Culture, Colorimetric Assay, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a, b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 30 minutes or 6 hours following treatment with 1 μM venetoclax or an equivalent volume of DMSO. Proteins were extracted from cell pellets and either separated by liquid chromatography and analyzed by tandem mass spectrometry for protein identification and quantification or enriched for phosphorylated species prior to LC–MS/MS analysis to profile phosphorylation-dependent signaling. Relative (a) global protein and (b) phosphoprotein abundance at 6 hours following venetoclax treatment in CD34-enriched and CD34-depleted populations were analyzed using proteomic network analysis.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Cell Culture, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Predicted transcription factor activity was evaluated in single-cell RNA-seq data presented in . Significantly enriched transcription factors were identified and denoted as enriched in progenitor (blue), monocytic (gold), or both (green) populations. Gray dots represent transcription factors that did not reach statistical significance (FDR < 0.05). b. Predicted activity of PU.1 visualized by each time point and drug condition. c. Pseudotime values generated in were used to determine each cell’s relative position along the myeloid differentiation trajectory. Predicted activity of PU.1 is visualized along its pseudotime trajectory in each drug condition. d. OCI-AML8227 cells were cultured with DMSO or 1 μM venetoclax and processed at either 0 or 72 hours for single-cell ATAC-seq. Differential peaks were identified from open chromatin regions in each condition. Significant peaks were compared between monocytic and progenitor clusters. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). e, f. Transcription factor motif analysis was performed on (e) upregulated and (f) downregulated regions in monocytic cells relative to progenitor cells as described in panel d. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). g. Venn diagram displaying the overlap of significantly dysregulated transcription factors identified from single-cell RNA-seq analysis in panel a compared to enriched motifs identified in panels e and f.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Activity Assay, RNA Sequencing, Generated, Cell Culture

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of the targeted CRISPR screen. A stable Cas9-expressing OCI-AML8227 cell line was generated by electroporation and sorted for GFP expression. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression. Image was created with BioRender. b. Live cell counts determined by forward/side scatter gating, excluding DAPI-stained cells. Counts were normalized to the average of their respective DMSO-treated controls. Blue lines and asterisks indicate comparisons with the safe-harbor locus AAVS1 (control) whereas black lines and asterisks indicate intra-sample comparisons. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. c–h. Live cell counts for each immunophenotypic population, normalized as described in panel b. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. i–j. Representative flow cytometry plots showing CD64-FITC and CD38-APC-Fire surface expression in OCI-AML8227 cells transduced with guide RNAs targeting AAVS1 or SPI1 following treatment with DMSO or 1 μM venetoclax. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: CRISPR, Expressing, Generated, Electroporation, Transduction, Cell Culture, Flow Cytometry, Staining, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression as described in . Quantification of total cell counts in additional knockout models of OCI-AML8227 cells. Total cell counts were normalized to the average of their respective DMSO-treated controls. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Sensitivity to venetoclax was unchanged by the experimental knockouts compared to control AAVS1 . b–g. Live cell counts for each immunophenotypic population were calculated by multiplying their proportions of live single cells from flow cytometric analysis by their normalized values in panel a. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Few changes were observed in knockouts relative to control. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Transduction, Electroporation, Cell Culture, Flow Cytometry, Expressing, Knock-Out, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. OCI-AML8227 cells were cultured in triplicate for 72 hours along an 8-point dose curve of DB2313. Cell viability was assessed by CellTiter Aqueous colorimetric assay. b. OCI-AML8227 cells were treated in triplicate with an 8×8 dose matrix of DB2313 and venetoclax for 72 hours prior to viability assessment by CellTiter Aqueous colorimetric assay. Zero interaction potency (ZIP) synergy scores were calculated on the average values for each drug dose. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. c–e. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 0.1 μM venetoclax, 0.5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Live cell counts were determined by forward/side scatter gating and exclusion of DAPI-stained cells, then normalized to DMSO-treated controls. Cells were analyzed by flow cytometry for CD34, CD38, CD64, and CD14 surface expression. Quantification of live cell counts for the remaining cell surface markers is shown in . Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. f–h. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 1 μM venetoclax, 5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Analysis was performed as described in panels c–e. i. Transcriptional signatures of nine primary AML samples selected for drug sensitivity evaluation are shown. One sample was excluded from downstream analyses due to widespread cell death (18-00105). j. Primary AML blasts from eight patients with stem-monocytic AML were cultured in triplicate for 72 hours along a 7-point dose curve with venetoclax, DB2313, or equimolar amounts of the drug combination. Viability was assessed using the Guava/EMD Millipore platform after a short incubation with Guava Nexin Reagent (Annexin V–PE + 7-AAD). ZIP synergy scores were calculated from averaged viability data across replicates for each drug dose in primary AML blasts shown in panel i. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. k. Venetoclax dose-response curves for each patient at a fixed dose of 1.25 μM DB2313, which corresponds to maximal synergy in panel j. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Cell Culture, Colorimetric Assay, Staining, Flow Cytometry, Expressing, Incubation