anti cd71 apc  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Schematic representation of the macrophage loading with HFt and subsequent transfer to cancer cells. Created in BioRender. Taciak, B. (2024) https://BioRender.com/s50b255 . b Snapshots from the movie recorded under the confocal microscopy (Supplementary Video ) in 15 min where BMDM macrophages loaded with HFt-FITC transfer it to EMT6 cancer cell labeled with red CellTrace. c Representative flow cytometry scatter plot of macrophages with internalized HFt-AF488 and MDA-MB-231 breast cancer cells labeled with CellTrace (CellTrace Far Red—APC). d and ( e ) Flow cytometry quantification of HFt-AF488 transfer from hMDM and (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various human cancer cell lines at 24 h following co-culture at 2:1 ratio (macrophages: cancer cells). Data are presented as mean ± SEM from n = 3 different donors (hMDM). f Representative confocal microscopy images of HFt-AF488 (green) transfer from hMDM (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to MDA‑MB‑231 breast cancer cells (blue) after 24 h of co-culture. Scale bar = 10 µm. Wheat Germ Agglutinin (WGA), Alexa Fluor-555 Conjugate (red) was used to visualize the cell membrane. g Comparative analysis of HFt-AF488 transfer from hMDM of different polarization states—M0, M1 (stimulated with LPS, 100 ng/ml), and M2 (stimulated with IL-4, 20 ng/ml)—to MDA-MB-231 breast cancer cells. hMDM were pre-loaded with HFt-AF488 (500 μg/ml) for 1 h at 37 °C, followed by co-culture with MDA-MB-231 cells for 4 and 24 h. Control conditions included co-cultures of M0, M1, and M2 hMDM with MDA-MB-231 cells without HFt-AF488. Data are presented as mean ± SEM from n = 3 different donors (hMDM). h and ( i ) Flow cytometry quantification of HFt-AF488 transfer from iPSC-derived macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various human cancer cell lines at 24 h following co-culture at 2:1 ratio (macrophages: cancer cells). Data are presented as mean ± SEM from n = 3 (A549, SK-OV-3) or n = 4 (DU145, MDA-MB-231, SW1353) independent replicates. j Representative confocal microscopy images of HFt-AF488 (green) transfer from iPSC-derived macrophages (loaded with HFt-AF-647 at concentration 500 µg/ml for 1 h at 37 °C) to MDA‑MB‑231 breast cancer cells (blue) after 24 h of co-culture. Scale bar = 10 µm. Wheat Germ Agglutinin (WGA), Alexa Fluor-555 Conjugate (red) was used to visualize the cell membrane. k Flow cytometry analysis of Tfn-AF647 transfer from RAW 264.7 and THP-1 macrophages (loaded with Tfn-AF-647 at concentration 0.2 mg/ml for 1 h at 37 °C) to cancer cells (EMT6 and MDA-MB-231, respectively) after 24 h of co-culture. Data are presented as mean ± SEM from n = 3 independent replicates. l Flow cytometry analysis of HFt-AF488 transfer from THP1-derived macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to various cancer cell lines after 4 h co-culture. Data are presented as mean ± SEM from n = 3 independent replicates. m Correlation of the percentage of HFt-AF488-positive recipient cells from human cell lines shown in ( l ) to their CD71 (TfR1) receptor expression assessed by flow cytometry. n Western blot analysis showing TfR1 gene knockdown efficiency in MDA-MB-231 cells using two different siRNA sequences and scramble as a control. o–q Flow cytometry analysis of AF488 fluorescence in MDA-MB-231 cancer cells with TfR1 gene knockdown using two different siRNA sequences, cells transfected with negative control (Scramble) siRNA or untreated cells (Control). Effect on ( o ) Tfn-AF488 and ( p ) HFt-AF488 uptake from medium and ( q ) HFt-AF488 transfer from THP-1 macrophages in 4 h co-culture was calculated relative to Scramble. Data are presented as mean ± SEM from n = 3 independent replicates. r and ( s ) Flow cytometry quantification of HFt-AF488 transfer from RAW 264.7 or THP-1 macrophages (loaded with HFt-AF488 at concentration 500 µg/ml for 1 h at 37 °C) to ( r ) EMT6 or ( s ) MDA-MB-231 breast cancer cells (respectively) at 24 h following direct, or Transwell membrane-separated co-culture system (macrophages seeded on Transwell insets). Co-culture of macrophages without HFt-AF488 and cancer cells [co-culture HFt(−)] was used as a control. Data are presented as mean ± SEM from n = 3 independent replicates. Statistical analysis was performed using one-way ANOVA with post-hoc Tukey HSD test. For all panels, * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: The cells were then incubated with one of the following antibodies: PE anti-human CD204/MSR1 antibody (1:20, 371904, BioLegend), APC anti-human CD71/TfR1 antibody (clone OKT9) (1:20, 17-0719-42, eBioscience), PE mouse IgG2a, κ isotype control antibody (1:20, 400214, BioLegend), APC mouse IgG1 κ isotype control antibody (P3.6.2.8.1) (1:20, 17-4714-82, eBioscience) for 1 h on ice in the dark.

Techniques: Confocal Microscopy, Labeling, Flow Cytometry, Concentration Assay, Co-Culture Assay, Membrane, Control, Derivative Assay, Expressing, Western Blot, Knockdown, Fluorescence, Transfection, Negative Control

Journal: Nucleic Acids Research

Article Title: The characteristics of CTCF binding sequences contribute to enhancer blocking activity

doi: 10.1093/nar/gkae666

Figure Lengend Snippet: An experimental system to evaluate the capacity of individual CTCF elements to block enhancer–promoter interactions. ( A ) Top, UCSC refseq genes at the genomic locus encompassing the alpha-globin locus. ATAC-seq track (black) shows the accessible regions with the alpha-globin enhancer-like elements (R1–R4) and genes ( Hba-a1 , Hba-a2 ) indicated. The blue track shows CTCF ChIP-seq with the peaks enriched over CTCF sites that are marked in red (forward orientation) and blue (reverse orientation) arrows as well as their corresponding label starting from the left with HS-94. Below a graphical representation of the alpha-globin locus indicating all three regulatory elements: CTCF sites in light blue and pink arrows, the enhancer-like elements in green, and the genes in red with the YFP tag in yellow and the red arrow indicating the transcriptionally active genes. The CTCF site of interest was inserted between the enhancer clusters and the promoters indicated by the blue star. An allele of the alpha-globin gene locus (117 kb) was hemizygously deleted in the reporter mouse ES cells (mESCs) as indicated. ( B ) The workflow starting with The CTCF site of interest inserted into the hemizygous alpha-globin locus of the reporter mESCs by the CRISPR-HDR method. The modified reporter mESCs were differentiated into EBs and the cells were harvested for analysis. The YFP level of the CD71+ cell population in the EBs were determined by FACS analysis and interpreted as the readout of the CTCF ability to block enhancer–promoter interaction as indicated. ( C ) Top panels, FACS plots to characterise the control E14 mESCs upon EB differentiation; left panel shows the proportion of CD71+ erythroid cells from the total EB population at day 7 of differentiation. Middle panel is a histogram showing the YFP signal ( Hba-a expression) specifically in the CD71+ erythroid population (as gated in left panel). The right panel shows the same data plotted for the whole population based on two parameters, the CD71 + erythroid marker and YFP. All CD71+ cells isolated from the differentiated EBs representing the erythroid population exhibit high level of YFP, indicating high expression of the alpha-globin as expected. The lower panels represent the same FACS profile for mESCs with the inserted HS-38R site. ( D ) RT-qPCR data showing that insertion of the HS-38 sequence in both the forward and reverse orientation (pink and maroon columns) significantly represses the expression of the alpha-globin genes ( Hba-a1/2 ) when compared to the ‘no insert’ and ‘negative insert’ controls. Data is normalised to the β -globin ( Hbb-b ). Bars indicate the standard deviation, the black dots represent single experiment, and the stars indicate the statistical significance resulting from unpaired, two-tailed t -tests, ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001. ( E ) Same as in (D) except this is based on the YFP levels measured by FACS of the reporter cells compared to the ‘no insert’ and ‘negative insert’ controls. ( F ) The expression of the alpha-globin genes is positively and significantly correlated to the level of the YFP in the samples (Linear regression, P < 0.0001, R 2 = 0.798).

Article Snippet: Differentiated EB cells were disaggregated and stained with the anti-mouse CD71-APC antibody (eBioscience, 11-0711-80) (1:8000 in staining buffer) and Hoechst (Invitrogen, 33258) (1:10 000 in staining buffer) for 30 min at 4°C in dark.

Techniques: Blocking Assay, ChIP-sequencing, CRISPR, Modification, Control, Expressing, Marker, Isolation, Quantitative RT-PCR, Sequencing, Standard Deviation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: The characteristics of CTCF binding sequences contribute to enhancer blocking activity

doi: 10.1093/nar/gkae666

Figure Lengend Snippet: Correlating binding activity and sequence characteristics with insulation function of individual CTCF elements. ( A ) There is no correlation between the core CTCF binding sequences (p-value of the FIMO analysis) with their ability to alter enhancer promoter activity (Spearman correlation, P = 0.65). ( B ) There is no correlation between the conservation of the inserted CTCF binding (PhastCons Vert30 genome conservation score) with their ability to block enhancer–promoter interaction (linear regression, P = 0.2171, R 2 = 0.0935). ( C ) There is no significant correlation between CTCF activity and CTCF enrichment in the mESCs (linear regression, P = 0.9379, R 2 = 0.0004). ( D ) There is also no correlation between CTCF activity and CTCF enrichment when only focused on the erythroid-specific alpha-globin CTCF binding sites in the CD71+ erythroid cells isolated from EBs (linear regression, P = 0.9243, R 2 = 0.0009). ( E ) CTCF binding sites with stronger enhancer–promoter blocking activity showed a higher level of Rad21 enrichment in mESCs (linear regression, P = 0.0416, R 2 = 0.2347). ( F ) The correlation between the activities of the alpha-globin locus CTCF binding sites and enrichment of cohesin in CD71+ cells isolated from the differentiated EBs was not statistically significant (linear regression, P = 0.1536, R 2 = 0.1925). ( G ) The presence of more than one overlapping core motif in the three tested CTCF sites (labeled as ‘multiple’) did not manifest as a stronger insulation effect as shown by the YFP levels when comparing the ‘multiple’ to ‘single’ (CTCF sites with only one core CTCF motif) datasets.

Article Snippet: Differentiated EB cells were disaggregated and stained with the anti-mouse CD71-APC antibody (eBioscience, 11-0711-80) (1:8000 in staining buffer) and Hoechst (Invitrogen, 33258) (1:10 000 in staining buffer) for 30 min at 4°C in dark.

Techniques: Binding Assay, Activity Assay, Sequencing, Insulation, Blocking Assay, Isolation, Labeling