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ATCC human leukemia 60 hl60 cells
Human Leukemia 60 Hl60 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hl60+cells/bio_rxiv__64898__2026__05__06__723311-186-7-12?v=ATCC
Average 99 stars, based on 6449 article reviews

human leukemia 60 hl60 cells - by Bioz Stars, 2026-07

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In vitro validation of the Pep@MNP platform for specific capture and identification of thyroid cancer cells. A and B, Immunofluorescence staining of the capture marker EpCAM ( A ) and the identification marker CKmix ( B ) in thyroid cancer cell lines (BHT101 and BCPAP) and a negative control cell line (HL60). Nuclei were counterstained with DAPI. Scale bars, 10 μm. C, Quantitative analysis of the MFI of CKmix in BHT101, BCPAP, and HL60 cells, demonstrating significantly higher expression in thyroid cancer cells. D and E, FCM analysis confirming the surface expression of EpCAM ( D ) and high intracellular expression of CKmix ( E ) in BHT101 and BCPAP cells but not in HL60 cells. F, Scanning electron microscopy images showing the specific binding of Pep@MNPs to target BCPAP cells, whereas bare MNPs show minimal interaction. Both nanoparticle types show negligible binding to the negative control HL60 cells. Scale bars, 5 μm (top row) and 3 μm (bottom row). G, Representative immunofluorescence images defining the criteria for identifying a captured BCPAP cell (CTCs-like phenotype: DAPI+/CKmix+/CD45 − ) and distinguishing it from a co-captured white blood cell (WBC; DAPI+/CKmix − /CD45 + ). H, Capture sensitivity analysis. The graph shows the capture efficiency of the platform for varying numbers of BCPAP cells spiked into a solution. I, Capture specificity analysis. The graph compares the capture efficiency of the platform for cells with high EpCAM expression (BCPAP), low EpCAM expression (BHT101), and negative EpCAM expression (HL60). ****, P value < 0.0001.

Journal: Clinical Cancer Research

Article Title: Clinical Significance for Risk Stratification of Papillary Thyroid Cancer by TUMORFISHER Circulating Tumor Cell Technique

doi: 10.1158/1078-0432.CCR-25-2694

Figure Lengend Snippet: In vitro validation of the Pep@MNP platform for specific capture and identification of thyroid cancer cells. A and B, Immunofluorescence staining of the capture marker EpCAM ( A ) and the identification marker CKmix ( B ) in thyroid cancer cell lines (BHT101 and BCPAP) and a negative control cell line (HL60). Nuclei were counterstained with DAPI. Scale bars, 10 μm. C, Quantitative analysis of the MFI of CKmix in BHT101, BCPAP, and HL60 cells, demonstrating significantly higher expression in thyroid cancer cells. D and E, FCM analysis confirming the surface expression of EpCAM ( D ) and high intracellular expression of CKmix ( E ) in BHT101 and BCPAP cells but not in HL60 cells. F, Scanning electron microscopy images showing the specific binding of Pep@MNPs to target BCPAP cells, whereas bare MNPs show minimal interaction. Both nanoparticle types show negligible binding to the negative control HL60 cells. Scale bars, 5 μm (top row) and 3 μm (bottom row). G, Representative immunofluorescence images defining the criteria for identifying a captured BCPAP cell (CTCs-like phenotype: DAPI+/CKmix+/CD45 − ) and distinguishing it from a co-captured white blood cell (WBC; DAPI+/CKmix − /CD45 + ). H, Capture sensitivity analysis. The graph shows the capture efficiency of the platform for varying numbers of BCPAP cells spiked into a solution. I, Capture specificity analysis. The graph compares the capture efficiency of the platform for cells with high EpCAM expression (BCPAP), low EpCAM expression (BHT101), and negative EpCAM expression (HL60). ****, P value < 0.0001.

Article Snippet: The human thyroid cancer cell lines BCPAP (RRID: CVCL_0153) and BHT101 (RRID: CVCL_1085), and the human promyelocytic leukemia cell line HL60 (RRID: CVCL_0002), were obtained from Procell Life Science & Technology in August 2021.

Techniques: In Vitro, Biomarker Discovery, Immunofluorescence, Staining, Marker, Negative Control, Expressing, Electron Microscopy, Binding Assay

The aryl hydrocarbon receptor (AhR) activation directly suppresses the NETosis. (a-f) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) in the present or absence of LPS (10 μg/mL) or PMA (50 nM). The release of dsDNA was stained, and measured using fluorescence microscope (scale bar: 50 μm) (a) and microplate reader (b, c) . The formation of NETs were detected by staining for NE/CitH3 (red), MPO (green) and DNA (blue) (scale bar: 20 μm) (d) . The levels of histone H4 (e) as well as the nucleus morphology (f) was detected (scale bar = 5 μm). (g) The HL60 cells were pre-transfected with shAhR, incubated with 1.25 % dimethyl sulfoxide, and treated with FICZ (100 nM) or DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The release of dsDNA was stained (scale bar: 50 μm). The data were presented as the means ± S.E.M. of three or five independent experiments. ## p < 0.01 vs. Control group; ** p < 0.01 vs. LPS or PMA group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Journal: Journal of Advanced Research

Article Title: Aryl hydrocarbon receptor impairs HK2-controlled flux of the hexosamine biosynthesis pathway to suppress NETosis in an N -glycosylation-dependent manner

doi: 10.1016/j.jare.2025.06.078

Figure Lengend Snippet: The aryl hydrocarbon receptor (AhR) activation directly suppresses the NETosis. (a-f) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) in the present or absence of LPS (10 μg/mL) or PMA (50 nM). The release of dsDNA was stained, and measured using fluorescence microscope (scale bar: 50 μm) (a) and microplate reader (b, c) . The formation of NETs were detected by staining for NE/CitH3 (red), MPO (green) and DNA (blue) (scale bar: 20 μm) (d) . The levels of histone H4 (e) as well as the nucleus morphology (f) was detected (scale bar = 5 μm). (g) The HL60 cells were pre-transfected with shAhR, incubated with 1.25 % dimethyl sulfoxide, and treated with FICZ (100 nM) or DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The release of dsDNA was stained (scale bar: 50 μm). The data were presented as the means ± S.E.M. of three or five independent experiments. ## p < 0.01 vs. Control group; ** p < 0.01 vs. LPS or PMA group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human promyelocytic leukaemia cell line HL60 was obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 20 % fetal bovine serum at 37 °C.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, Transfection, Incubation, Control

The aryl hydrocarbon receptor (AhR) activation inhibits NETosis with the help of neutrophil elastase (NE). (a) The HL60 cells were pre-transfected with pcDNA-NE or pcDNA-PAD4 , differentiated for 5 days by incubation of 1.25 % dimethyl sulfoxide (DMSO), and treated with FICZ (100 nM) or DIM (10 μM) at the present or absence of lipopolysaccharide (LPS, 10 μg/mL) or phorbol-12-myristate-13-acetate (PMA, 50 nM). The release of dsDNA was stained (scale bar: 50 μm). (b-d) The HL60 cells were differentiated for 0, 1, 3 and 5 days, and incubated with 1.25 % DMSO in the presence of FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM). The mRNA level of NE (b) and protein level of CTSC (c, d) was detected. ( e-h) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM), and the protein level (e) and activity (f) of NE was detected. The NE (red) and DNA (blue) was stained for detecting the nuclear localisation of NE (scale bar = 20 μm) (g) . The NE (green) and F-actin (red) was stained for detecting the cutting capacity of NE (scale bar: 5 μm) (h) . (i) The HL60 cells were pre-transfected with shAhR, incubated with 1.25 % DMSO, and treated with FICZ (100 nM) and DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The NE activity was determined. The data were presented as the means ± S.E.M. of three or five independent experiments. ## p < 0.01 vs. Control group; ** p < 0.01 vs. LPS or PMA group; $$ p < 0.01 vs. LPS/PMA + FICZ group; ++ p < 0.01 vs. LPS/PMA + DIM group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Journal: Journal of Advanced Research

Article Title: Aryl hydrocarbon receptor impairs HK2-controlled flux of the hexosamine biosynthesis pathway to suppress NETosis in an N -glycosylation-dependent manner

doi: 10.1016/j.jare.2025.06.078

Figure Lengend Snippet: The aryl hydrocarbon receptor (AhR) activation inhibits NETosis with the help of neutrophil elastase (NE). (a) The HL60 cells were pre-transfected with pcDNA-NE or pcDNA-PAD4 , differentiated for 5 days by incubation of 1.25 % dimethyl sulfoxide (DMSO), and treated with FICZ (100 nM) or DIM (10 μM) at the present or absence of lipopolysaccharide (LPS, 10 μg/mL) or phorbol-12-myristate-13-acetate (PMA, 50 nM). The release of dsDNA was stained (scale bar: 50 μm). (b-d) The HL60 cells were differentiated for 0, 1, 3 and 5 days, and incubated with 1.25 % DMSO in the presence of FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM). The mRNA level of NE (b) and protein level of CTSC (c, d) was detected. ( e-h) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM), and the protein level (e) and activity (f) of NE was detected. The NE (red) and DNA (blue) was stained for detecting the nuclear localisation of NE (scale bar = 20 μm) (g) . The NE (green) and F-actin (red) was stained for detecting the cutting capacity of NE (scale bar: 5 μm) (h) . (i) The HL60 cells were pre-transfected with shAhR, incubated with 1.25 % DMSO, and treated with FICZ (100 nM) and DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The NE activity was determined. The data were presented as the means ± S.E.M. of three or five independent experiments. ## p < 0.01 vs. Control group; ** p < 0.01 vs. LPS or PMA group; $$ p < 0.01 vs. LPS/PMA + FICZ group; ++ p < 0.01 vs. LPS/PMA + DIM group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques: Activation Assay, Transfection, Incubation, Staining, Activity Assay, Control

The aryl hydrocarbon receptor (AhR) activation prevents the N- glycosylation of alpha-1 antitrypsin (AAT) and alpha-2-macroglobulin (A2M) to down-regulate the neutrophil elastase (NE) activity. (a) The recombinant protein of NE was incubated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM), and NE activity was determined. (b, c) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of lipopolysaccharide (LPS, 10 μg/mL) or phorbol-12-myristate-13-acetate (PMA, 50 nM). The cytoplasmic proteins in each group were isolated and co-incubated with the recombinant protein of NE, and NE activity was determined (b) . The association of AAT, A2M or secretory leukocyte protease inhibitor (SLPI) with NE was detected (c) . (d) The HL60 cells were pre-transfected with shAAT or shA2M, incubated with 1.25 % dimethyl sulfoxide, treated with FICZ (100 nM) and DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM), and NE activity was determined. (e-h) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The levels of AAT, A2M, SLPI (e) , ROS (f) , N- glycosylation (g) and neutral lipid content (h) were detected. ( i, j) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM), and tunicamycin (TM; 1 μM) or MK-8719 (21 nM) were jointly given at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The association of AAT or A2M with NE (i) and release of dsDNA (scale bar: 50 μm) (j) were detected. The data were presented as the means ± S.E.M. of three or five independent experiments. # p < 0.05, ## p < 0.01 vs. Control group; * p < 0.05, ** p < 0.01 vs. LPS group; $$ p < 0.01 vs. LPS + FICZ group; ++ p < 0.01 vs. LPS + DIM or CH223191 group.

Journal: Journal of Advanced Research

Article Title: Aryl hydrocarbon receptor impairs HK2-controlled flux of the hexosamine biosynthesis pathway to suppress NETosis in an N -glycosylation-dependent manner

doi: 10.1016/j.jare.2025.06.078

Figure Lengend Snippet: The aryl hydrocarbon receptor (AhR) activation prevents the N- glycosylation of alpha-1 antitrypsin (AAT) and alpha-2-macroglobulin (A2M) to down-regulate the neutrophil elastase (NE) activity. (a) The recombinant protein of NE was incubated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM), and NE activity was determined. (b, c) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of lipopolysaccharide (LPS, 10 μg/mL) or phorbol-12-myristate-13-acetate (PMA, 50 nM). The cytoplasmic proteins in each group were isolated and co-incubated with the recombinant protein of NE, and NE activity was determined (b) . The association of AAT, A2M or secretory leukocyte protease inhibitor (SLPI) with NE was detected (c) . (d) The HL60 cells were pre-transfected with shAAT or shA2M, incubated with 1.25 % dimethyl sulfoxide, treated with FICZ (100 nM) and DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM), and NE activity was determined. (e-h) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The levels of AAT, A2M, SLPI (e) , ROS (f) , N- glycosylation (g) and neutral lipid content (h) were detected. ( i, j) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM), and tunicamycin (TM; 1 μM) or MK-8719 (21 nM) were jointly given at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The association of AAT or A2M with NE (i) and release of dsDNA (scale bar: 50 μm) (j) were detected. The data were presented as the means ± S.E.M. of three or five independent experiments. # p < 0.05, ## p < 0.01 vs. Control group; * p < 0.05, ** p < 0.01 vs. LPS group; $$ p < 0.01 vs. LPS + FICZ group; ++ p < 0.01 vs. LPS + DIM or CH223191 group.

Techniques: Activation Assay, Glycoproteomics, Activity Assay, Recombinant, Incubation, Isolation, Protease Inhibitor, Transfection, Control

The aryl hydrocarbon receptor (AhR) acts as an E3 ligase to promote the ubiquitination of hexokinase-2 (HK2) and restrain glucose metabolism. (a-c) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of lipopolysaccharide (LPS, 10 μg/mL) or phorbol-12-myristate-13-acetate (PMA, 50 nM). The relative activity (a) as well as protein levels ( b, c ) of hexokinase 2 (HK2) and phosphofructokinase (PFK) were detected. (d-f) The HL60 cells were pre-transfected with pcDNA -HK2, incubated with 1.25 % dimethyl sulfoxide (DMSO), and treated with FICZ (100 nM) and DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The activity of NE (d) and levels of UDP-GlcNAc (e) , HK2, PFK (f) were detected. (g) The neutrophils were treated with FICZ (100 nM) or DIM (10 μM) before receiving LPS (10 μg/mL) and CHX (15 μg/mL) for 0, 0.5, 1, 1.5 h, and the protein level of HK2 was detected. (h, i) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM), and MG132 (25 μg/mL) or hydroxychloroquine (HCQ; 10 μM) were jointly given at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The protein level (h) and ubiquitination (i) of HK2 was detected. (j, k) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL). The co-localization of HK2 and PSMD2 was stained (scale bar: 5 μm) (j) . The mRNA levels of CYP1A1 and CYP1B1 was detected (k) . (l) The HL60 cells were pre-transfected with shARNT, incubated with 1.25 % DMSO, treated with FICZ (100 nM) or DIM (10 μM) at the present or absence of LPS (10 μg/mL), and release of dsDNA was stained (scale bar = 50 μm). (m, n) The association of AhR and HK2 was simulated using molecular docking and the association of AhR and HK2 was detected. (o) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL), and the association of ubiquitination at Lys 48 and Lys 63 of HK2 protein was detected. The data were presented as the means ± S.E.M. of three or five independent experiments. # p < 0.05, ## p < 0.01 vs. Control group; * p < 0.05, ** p < 0.01 vs. LPS group; $ p < 0.05, $$ p < 0.01 vs. LPS + FICZ group; + p < 0.01, ++ p < 0.01 vs. LPS + DIM group.

Journal: Journal of Advanced Research

Article Title: Aryl hydrocarbon receptor impairs HK2-controlled flux of the hexosamine biosynthesis pathway to suppress NETosis in an N -glycosylation-dependent manner

doi: 10.1016/j.jare.2025.06.078

Figure Lengend Snippet: The aryl hydrocarbon receptor (AhR) acts as an E3 ligase to promote the ubiquitination of hexokinase-2 (HK2) and restrain glucose metabolism. (a-c) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of lipopolysaccharide (LPS, 10 μg/mL) or phorbol-12-myristate-13-acetate (PMA, 50 nM). The relative activity (a) as well as protein levels ( b, c ) of hexokinase 2 (HK2) and phosphofructokinase (PFK) were detected. (d-f) The HL60 cells were pre-transfected with pcDNA -HK2, incubated with 1.25 % dimethyl sulfoxide (DMSO), and treated with FICZ (100 nM) and DIM (10 μM) at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The activity of NE (d) and levels of UDP-GlcNAc (e) , HK2, PFK (f) were detected. (g) The neutrophils were treated with FICZ (100 nM) or DIM (10 μM) before receiving LPS (10 μg/mL) and CHX (15 μg/mL) for 0, 0.5, 1, 1.5 h, and the protein level of HK2 was detected. (h, i) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM), and MG132 (25 μg/mL) or hydroxychloroquine (HCQ; 10 μM) were jointly given at the present or absence of LPS (10 μg/mL) or PMA (50 nM). The protein level (h) and ubiquitination (i) of HK2 was detected. (j, k) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL). The co-localization of HK2 and PSMD2 was stained (scale bar: 5 μm) (j) . The mRNA levels of CYP1A1 and CYP1B1 was detected (k) . (l) The HL60 cells were pre-transfected with shARNT, incubated with 1.25 % DMSO, treated with FICZ (100 nM) or DIM (10 μM) at the present or absence of LPS (10 μg/mL), and release of dsDNA was stained (scale bar = 50 μm). (m, n) The association of AhR and HK2 was simulated using molecular docking and the association of AhR and HK2 was detected. (o) The neutrophils were treated with FICZ (100 nM), DIM (10 μM) or CH223191 (10 μM) at the present or absence of LPS (10 μg/mL), and the association of ubiquitination at Lys 48 and Lys 63 of HK2 protein was detected. The data were presented as the means ± S.E.M. of three or five independent experiments. # p < 0.05, ## p < 0.01 vs. Control group; * p < 0.05, ** p < 0.01 vs. LPS group; $ p < 0.05, $$ p < 0.01 vs. LPS + FICZ group; + p < 0.01, ++ p < 0.01 vs. LPS + DIM group.

Techniques: Ubiquitin Proteomics, Activity Assay, Transfection, Incubation, Staining, Control

CD45 was knocked down by CRISPR/Cas9 in the human promyelocytic cell line HL60 (CD45KO). (A) Lysates from HL60, SCR HL60 or CD45KO HL60 cells differentiated into a neutrophil like state were immunoblotted for CD45 or Gapdh. (B) Densitometry analysis revealed a <98% reduction in CD45 expression (normalized to Gapdh) in CD45KO HL60s relative to non-transduced HL60 cells or SCR HL60 cells. Data is expressed as mean ± SEM normalized to Gapdh and relative to non-transduced HL60 cells (n = 3, **** p<0.0001). (C) Flow cytometry analysis using a PerCP labeled anti-CD45 mAb (30-F11), and a PerCP labeled IgG matched control mAb to show non-specific background fluorescence, revealed a <98% reduction in CD45 surface expression in differentiated CD45KO HL60 cells relative to SCR HL60 cells. Data is mean fluorescence intensity normalized to HL60 Scr IgG control and is expressed as mean ± SEM (n =3, ****, p<0.0001). (E,F) Differentiated SCR HL60 cells and CD45KO HL60 cells were stimulated with 1.25μM LaB and 5μM fMLF to induce degranulation before assessment of surface expression of CD63 by flow cytometry. Data shown are fold-change in mean fluorescence intensity (MFI) normalized to SCR HL60 IgG and are expressed as mean ± SEM (n=3, ** p<0.01). (G,H) Differentiated SCR HL60 and CD45KO HL60 cells were stimulated with 100nM fMLF for 60 minutes at 37°C before fluorescent microsphere phagocytosis/uptake was quantified by measuring changes in fluorescence by flow cytometry. Data is mean fluorescence intensity normalized to SCR HL60 and is expressed as mean ± SEM (n=5, ** p<0.01).

Journal: bioRxiv

Article Title: The Protein Tyrosine Phosphatase CD45 promotes PMN Transepithelial Migration, Antimicrobial Function and Colonic Mucosal Repair

doi: 10.64898/2026.03.25.714205

Figure Lengend Snippet: CD45 was knocked down by CRISPR/Cas9 in the human promyelocytic cell line HL60 (CD45KO). (A) Lysates from HL60, SCR HL60 or CD45KO HL60 cells differentiated into a neutrophil like state were immunoblotted for CD45 or Gapdh. (B) Densitometry analysis revealed a <98% reduction in CD45 expression (normalized to Gapdh) in CD45KO HL60s relative to non-transduced HL60 cells or SCR HL60 cells. Data is expressed as mean ± SEM normalized to Gapdh and relative to non-transduced HL60 cells (n = 3, **** p<0.0001). (C) Flow cytometry analysis using a PerCP labeled anti-CD45 mAb (30-F11), and a PerCP labeled IgG matched control mAb to show non-specific background fluorescence, revealed a <98% reduction in CD45 surface expression in differentiated CD45KO HL60 cells relative to SCR HL60 cells. Data is mean fluorescence intensity normalized to HL60 Scr IgG control and is expressed as mean ± SEM (n =3, ****, p<0.0001). (E,F) Differentiated SCR HL60 cells and CD45KO HL60 cells were stimulated with 1.25μM LaB and 5μM fMLF to induce degranulation before assessment of surface expression of CD63 by flow cytometry. Data shown are fold-change in mean fluorescence intensity (MFI) normalized to SCR HL60 IgG and are expressed as mean ± SEM (n=3, ** p<0.01). (G,H) Differentiated SCR HL60 and CD45KO HL60 cells were stimulated with 100nM fMLF for 60 minutes at 37°C before fluorescent microsphere phagocytosis/uptake was quantified by measuring changes in fluorescence by flow cytometry. Data is mean fluorescence intensity normalized to SCR HL60 and is expressed as mean ± SEM (n=5, ** p<0.01).

Article Snippet: Human myelomonocytic HL60 cells were obtained from ATCC (clone#CCL-240).

Techniques: CRISPR, Expressing, Flow Cytometry, Labeling, Control, Fluorescence

(A,B) Human PMN exposed to 500nM CD45 inhibitor VI or vehicle control were incubated with a FITC conjugated anti-CD11b mAb or a FITC conjugated isotype matched IgG control mAb and assessed by flow cytometry. Inhibition of CD45 phosphatase activity resulted in a significant decrease in levels of CD11b surface expression relative to vehicle control (Vehicle). Data ± SEM represent fold change in mean fluorescence intensity for CD11b normalized to Ctrl (n=3, * p<0.05, ** p<0.01). (C,D) Human PMN exposed to 500nM CD45 inhibitor VI or vehicle control were assessed for CD11b activation by flow cytometry using the activation reporter antibody CBRM1/5. Inhibition of CD45 phosphatase activity resulted in a significant decrease in levels of active CD11b on the surface of PMNs. Data ± SEM represent fold change in mean fluorescence intensity of CBRM1/5 normalized to Ctrl (n=3, ** p<0.01). (E,F) Decreased surface expression of CD11b was detected by flow cytometry of CD45KO HL60 cells relative to SCR HL60s or non-transduced HL60 cells. Data ± SEM represent fold change in mean fluorescence intensity of CD11b normalized to non-transduced HL60 cells (n=3, * p<0.05). (G,H) Decreased surface expression of active CD11b in CD45KO HL60 cells relative to SCR HL60s or non-transduced HL60 cells was detected by flow cytometry. Data ± SEM represent fold change in mean fluorescence intensity of CBRM1/5 normalized to non-transduced HL60 cells (n=3, * p<0.05). (I,J) PMN isolated from WT mice were exposed to 500nM CD45 inhibitor VI or vehicle control and incubated with an APC conjugated anti-CD11b mAb or an APC conjugated isotype matched control mAb before analysis of CD11b surface expression by flow cytometry. Inhibition of CD45 phosphatase activity resulted in a significant decrease in levels of CD11b surface expression. Data ± SEM represent fold change in mean fluorescence intensity for CD11b normalized to vehicle control (n=4, * p<0.05). (K,L) PMN isolated from MRP8-Cre;Cd45 fl/fl mice or Cd45 fl/fl mice were incubated with an APC conjugated anti-CD11b mAb or an APC conjugated isotype matched control mAb before analysis of CD11b surface expression by flow cytometry. Data ± SEM represent fold change in mean fluorescence intensity of CD11b normalized to Cd45 fl/fl PMN (n=4, ** p<0.01).

Journal: bioRxiv

Article Title: The Protein Tyrosine Phosphatase CD45 promotes PMN Transepithelial Migration, Antimicrobial Function and Colonic Mucosal Repair

doi: 10.64898/2026.03.25.714205

Figure Lengend Snippet: (A,B) Human PMN exposed to 500nM CD45 inhibitor VI or vehicle control were incubated with a FITC conjugated anti-CD11b mAb or a FITC conjugated isotype matched IgG control mAb and assessed by flow cytometry. Inhibition of CD45 phosphatase activity resulted in a significant decrease in levels of CD11b surface expression relative to vehicle control (Vehicle). Data ± SEM represent fold change in mean fluorescence intensity for CD11b normalized to Ctrl (n=3, * p<0.05, ** p<0.01). (C,D) Human PMN exposed to 500nM CD45 inhibitor VI or vehicle control were assessed for CD11b activation by flow cytometry using the activation reporter antibody CBRM1/5. Inhibition of CD45 phosphatase activity resulted in a significant decrease in levels of active CD11b on the surface of PMNs. Data ± SEM represent fold change in mean fluorescence intensity of CBRM1/5 normalized to Ctrl (n=3, ** p<0.01). (E,F) Decreased surface expression of CD11b was detected by flow cytometry of CD45KO HL60 cells relative to SCR HL60s or non-transduced HL60 cells. Data ± SEM represent fold change in mean fluorescence intensity of CD11b normalized to non-transduced HL60 cells (n=3, * p<0.05). (G,H) Decreased surface expression of active CD11b in CD45KO HL60 cells relative to SCR HL60s or non-transduced HL60 cells was detected by flow cytometry. Data ± SEM represent fold change in mean fluorescence intensity of CBRM1/5 normalized to non-transduced HL60 cells (n=3, * p<0.05). (I,J) PMN isolated from WT mice were exposed to 500nM CD45 inhibitor VI or vehicle control and incubated with an APC conjugated anti-CD11b mAb or an APC conjugated isotype matched control mAb before analysis of CD11b surface expression by flow cytometry. Inhibition of CD45 phosphatase activity resulted in a significant decrease in levels of CD11b surface expression. Data ± SEM represent fold change in mean fluorescence intensity for CD11b normalized to vehicle control (n=4, * p<0.05). (K,L) PMN isolated from MRP8-Cre;Cd45 fl/fl mice or Cd45 fl/fl mice were incubated with an APC conjugated anti-CD11b mAb or an APC conjugated isotype matched control mAb before analysis of CD11b surface expression by flow cytometry. Data ± SEM represent fold change in mean fluorescence intensity of CD11b normalized to Cd45 fl/fl PMN (n=4, ** p<0.01).

Article Snippet: Human myelomonocytic HL60 cells were obtained from ATCC (clone#CCL-240).

Techniques: Control, Incubation, Flow Cytometry, Inhibition, Activity Assay, Expressing, Fluorescence, Activation Assay, Isolation

(A) CD45KO HL60 cells or SCR control HL60 cells differentiated to a PMN like state were stimulated with 100nM fMLF over 60 minutes. Representative blots are shown for indicated proteins or phosphoproteins. Blots are representative of n=3 independent experiments. (B) Densitometry showing statistically significant decreases in Lyn dephosphorylation at Tyr 507 but no changes in Hck or Lyn phosphorylation upon CD45 knockdown. Data ± SEM represent phosphorylated protein levels normalized to GAPDH relative to total protein levels normalized to GAPDH (n=3, ** p<0.01; *** p<0.001; **** p<0.0001). (C) PMN from MRP8-Cre; Cd45 fl/fl mice and Cd45 fl/fl mice were stimulated with 100nM fMLF over a 1-hour time course. Representative blots are shown for indicated proteins. Blots are representative of n=3 independent experiments. (D) Densitometry showing statistically significant decreases in Lyn dephosphorylation at Tyr 507 but no changes in Hck or Lyn phosphorylation in PMN from MRP8-Cre; Cd45 fl/fl mice relative to PMN from Cd45 fl/fl mice. Data ± SEM represent phosphorylated protein levels normalized to GAPDH relative to total protein levels normalized to GAPDH (n=3, * p<0.05; ** p<0.01; *** p<0.001).

Journal: bioRxiv

Article Title: The Protein Tyrosine Phosphatase CD45 promotes PMN Transepithelial Migration, Antimicrobial Function and Colonic Mucosal Repair

doi: 10.64898/2026.03.25.714205

Figure Lengend Snippet: (A) CD45KO HL60 cells or SCR control HL60 cells differentiated to a PMN like state were stimulated with 100nM fMLF over 60 minutes. Representative blots are shown for indicated proteins or phosphoproteins. Blots are representative of n=3 independent experiments. (B) Densitometry showing statistically significant decreases in Lyn dephosphorylation at Tyr 507 but no changes in Hck or Lyn phosphorylation upon CD45 knockdown. Data ± SEM represent phosphorylated protein levels normalized to GAPDH relative to total protein levels normalized to GAPDH (n=3, ** p<0.01; *** p<0.001; **** p<0.0001). (C) PMN from MRP8-Cre; Cd45 fl/fl mice and Cd45 fl/fl mice were stimulated with 100nM fMLF over a 1-hour time course. Representative blots are shown for indicated proteins. Blots are representative of n=3 independent experiments. (D) Densitometry showing statistically significant decreases in Lyn dephosphorylation at Tyr 507 but no changes in Hck or Lyn phosphorylation in PMN from MRP8-Cre; Cd45 fl/fl mice relative to PMN from Cd45 fl/fl mice. Data ± SEM represent phosphorylated protein levels normalized to GAPDH relative to total protein levels normalized to GAPDH (n=3, * p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: Human myelomonocytic HL60 cells were obtained from ATCC (clone#CCL-240).

Techniques: Control, De-Phosphorylation Assay, Phospho-proteomics, Knockdown

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