Journal: Scientific Reports

Article Title: Detection of CLCF1 protein expression by flow cytometry

doi: 10.1038/s41598-024-64101-9

Figure Lengend Snippet: CLCF1 can be detected by flow cytometry in Ba/F3 cells transduced with human and mouse CLCF1 cDNA. Ba/F3 cells were transduced with empty pMX retroviruses or pMX coding for protein C epitope-tagged human or mouse CLCF1 cDNA. ( a ) hCLCF1 expression was confirmed by Western Blot using mAbs specific for the tag (anti-ProtC) or for CLCF1 (anti-CLCF1). Ba/F3 cells transduced with empty pMX retroviruses (Ba/F3 empty vector) and recombinant hCLCF1 produced in E. coli (10 ng) were used as controls. The molecular weight difference between the recombinant hCLCF1 and the hCLCF1 from Ba/F3 cells is seemingly due to post-transcriptional modifications in eukaryote cells. Western Blot images were cropped. Original blots are presented in Fig. . (n = 5) ( b – d ) Cells were fixed and permeabilized with formaldehyde and methanol. Flow cytometry fluorescence signal in control (filled grey histograms) or hCLCF1 (black line histograms) expressing Ba/F3 cells was obtained using ( b ) indirect detection with an anti-CLCF1 mAb followed by an Alexa Fluor 488-conjugated secondary antibody (n = 4) or ( c , d ) direct detection with an anti-CLCF1 mAb conjugated to ( c ) CF405M (n = 7) or ( d ) Alexa Fluor 647 (n = 5). Control stainings with secondary antibodies and/or unstained cells are presented in Fig. a–c. ( e ) Cells were fixed and permeabilized with formaldehyde and methanol (left panel), the eBioscience FoxP3/Transcription factor staining buffer set (middle panel) or the BD Cytofix/Cytoperm fixation/permeabilization kit (right panel). Flow cytometry fluorescence signal was detected in control (filled grey histogram) or hCLCF1 expressing (black line histogram) Ba/F3 cells stained with an Alexa Fluor 647-conjugated anti-CLCF1 mAb. (n = 3) ( f ) Cells were fixed and permeabilized with formaldehyde and methanol. Flow cytometry fluorescence signal was detected in control (filled grey histogram), human (dotted line histogram) or mouse (solid line histogram) CLCF1 expressing Ba/F3 cells that were stained with a CF405M-conjugated anti-CLCF1 mAb. (n = 3).

Article Snippet: Codon optimized synthetic cDNAs coding for human CLCF1 tagged with the protein C epitope (EDQVDPRLIDGK), human CRLF1-T2A-CLCF1 , mouse CLCF1, human CRLF1-SGGG linker-GPI anchor (CNTFR a.a 346-372) fusion protein, and human CNTFRα were generated by GeneArt (Thermo Fisher Scientific, Burlington ON).

Techniques: Flow Cytometry, Transduction, Expressing, Western Blot, Plasmid Preparation, Recombinant, Produced, Molecular Weight, Fluorescence, Control, Staining

Journal: Scientific Reports

Article Title: Detection of CLCF1 protein expression by flow cytometry

doi: 10.1038/s41598-024-64101-9

Figure Lengend Snippet: The anti-CLCF1 mAb recognizes an epitope that encompasses CLCF1 binding site III and can inhibit CLCF1-CRLF1 interaction. ( a ) Ba/F3 cells were transduced with empty pMX retroviruses (empty vector; filled grey histogram) or pMX coding for human CLCF1 (hCLCF1; dotted line histogram) or human CLCF1 and CRLF1 (hCRLF1-T2A-hCLCF1; solid line histogram). CRLF1-induced CLCF1 secretion was inhibited by adding brefeldin A for 4 h and cells were fixed and permeabilized with formaldehyde and methanol. Flow cytometry fluorescence signal was detected with a CF405M-conjugated anti-CLCF1 mAb. (n = 4) ( b ) Recombinant wild-type CLCF1 (hCLCF1), binding site I mutant CLCF1 (hCLCF1 W67A ) or binding site III mutant CLCF1 (hCLCF1 F151A/K154A ) were produced in E. coli and subjected to Western Blot analysis using anti-ProtC (upper panel) or anti-CLCF1 (lower panel) mAbs. Western Blot images were cropped. Original blots are presented in Fig. . (n = 3) ( c ) Ba/F3 cells were transduced with a pMX derivative coding for a hCRLF1-sggg linker-GPI anchor fusion protein and incubated with biotinylated hCLCF1 in the presence of anti-CLCF1 mAb (solid line histogram) or isotype control (dotted line histogram). Binding of CLCF1 to cell surface-bound CRLF1 was detected using PE-conjugated streptavidin. Fluorescence signal of PE-conjugated streptavidin background binding in the absence of hCLCF1 is used as a negative control (filled grey histogram). Bar graphs represent mean fluorescence intensity (MFI) of PE-conjugated streptavidin ± SEM. Statistical significance was assessed using an ANOVA with a Bonferroni post-hoc test, with **p < 0.01 and ****p < 0.0001. (n = 6).

Article Snippet: Codon optimized synthetic cDNAs coding for human CLCF1 tagged with the protein C epitope (EDQVDPRLIDGK), human CRLF1-T2A-CLCF1 , mouse CLCF1, human CRLF1-SGGG linker-GPI anchor (CNTFR a.a 346-372) fusion protein, and human CNTFRα were generated by GeneArt (Thermo Fisher Scientific, Burlington ON).

Techniques: Binding Assay, Transduction, Plasmid Preparation, Flow Cytometry, Fluorescence, Recombinant, Mutagenesis, Produced, Western Blot, Incubation, Control, Negative Control

Journal: Scientific Reports

Article Title: Detection of CLCF1 protein expression by flow cytometry

doi: 10.1038/s41598-024-64101-9

Figure Lengend Snippet: The anti-CLCF1 mAb can inhibit CLCF1 biological activity in vitro . Ba/F3 cells were transduced with pMX retroviruses coding for the three receptor chains (CNTFRα, LIFRβ and gp130) of the CNTF receptor (Ba/F3 CNTFR). ( a ) Ba/F3 CNTFR triplicate cell cultures were stimulated with hCLCF1 (25 ng/ml) in the presence of vehicle, isotype control or anti-CLCF1 mAb (2.5μg/ml). Cell lysates were subjected to Western Blot analysis with mAbs specific for phosphorylated (upper panel) or total STAT3 (lower panel). Western Blot signal intensity was quantified using the ImageJ software. Western Blot images were cropped. Original blots are presented in Fig. . Bar graph represents pSTAT3 signal intensity ± SEM. Statistical significance was assessed using an ANOVA with a Bonferroni post-hoc test, with ***p < 0.001 and ****p < 0.0001. (n = 3) ( b ) Proliferation of Ba/F3 CNTFR cells in response to increasing concentrations of hCLCF1 was assessed in the presence of 2.5 μg/ml of anti-CLCF1 mAb (red triangles), isotype control (black squares) or vehicle (grey circles). Ba/F3 proliferation was measured in triplicates using an alamarBlue fluorometric assay. Statistical significance was assessed using a one-way ANOVA and a Tukey post-hoc test for each concentration point of hCLCF1, with *p < 0.05 and **p < 0.01. (n = 5).

Article Snippet: Codon optimized synthetic cDNAs coding for human CLCF1 tagged with the protein C epitope (EDQVDPRLIDGK), human CRLF1-T2A-CLCF1 , mouse CLCF1, human CRLF1-SGGG linker-GPI anchor (CNTFR a.a 346-372) fusion protein, and human CNTFRα were generated by GeneArt (Thermo Fisher Scientific, Burlington ON).

Techniques: Activity Assay, In Vitro, Transduction, Control, Western Blot, Software, Concentration Assay

Journal: Scientific Reports

Article Title: Detection of CLCF1 protein expression by flow cytometry

doi: 10.1038/s41598-024-64101-9

Figure Lengend Snippet: CLCF1 is expressed by mouse CD4 + and CD8 + T cells. Mouse splenocytes were analysed ( a ) upon isolation or ( b ) after 3 d of activation with plate-bound anti-TCR and soluble anti-CD28. ( a , b ) All cells were stimulated with PMA/ionomycin/brefeldin A for 4 h, and fixed and permeabilized with the eBioscience FoxP3/Transcription factor staining buffer set. The panels on the left show the immune population investigated (top : CD3 + ; middle and bottom : CD4 + and CD8 + gated on CD3 + cells). The panels to the right compare the fluorescence signal of the Alexa fluor 647-conjugated anti-CLCF1 mAb of cells isolated from Clcf1 knock-out mice (KO; filled grey histogram) or wild-type mice (WT; black line histogram). The bar graphs show the relative fluorescence intensity (RFI) for the Alexa Fluor 647-conjugated anti-CLCF1 mAb ± SEM. Statistical significance was assessed using Student’s t test, with *p < 0.05, **p < 0.01, and ****p < 0,0001. Control unstained cells are presented in Fig. d,e. Experiments were performed in triplicate, and replicated in three separate experiments.

Article Snippet: Codon optimized synthetic cDNAs coding for human CLCF1 tagged with the protein C epitope (EDQVDPRLIDGK), human CRLF1-T2A-CLCF1 , mouse CLCF1, human CRLF1-SGGG linker-GPI anchor (CNTFR a.a 346-372) fusion protein, and human CNTFRα were generated by GeneArt (Thermo Fisher Scientific, Burlington ON).

Techniques: Isolation, Activation Assay, Staining, Fluorescence, Knock-Out, Control

Journal: Scientific Reports

Article Title: Detection of CLCF1 protein expression by flow cytometry

doi: 10.1038/s41598-024-64101-9

Figure Lengend Snippet: CLCF1 is expressed by differentiated Th1 cells. Mouse splenic CD4 + T cells were activated with plate-bound anti-TCR and soluble anti-CD28 alone (Th0) or supplemented with neutralizing anti-IL-4 mAb and mouse IL-12 (10 ng/ml) (Th1) for 5 d. Cells were re-stimulated with PMA/ionomycin/brefeldin A for 4 h, and fixed and permeabilized with the eBioscience FoxP3/Transcription factor staining buffer set. A flow cytometry histogram graph and a bar graph are shown for each stimulation condition (Left : Th0; Right : Th1). The flow cytometry histogram graph compares the fluorescence signal of the Alexa Fluor 647-conjugated anti-CLCF1 mAb of cells isolated from a Clcf1 KO (grey filled histogram) or WT (black line histogram) mouse. The bar graph represents relative fluorescence intensity (RFI) of the Alexa Fluor 647-conjugated anti-CLCF1 mAb ± SEM. Statistical significance was assessed using Student’s t test, with **p < 0.01 and ****p < 0.0001. Control unstained cells are presented in Fig. f. The experiment was performed in triplicate, and replicated in three separate experiments.

Article Snippet: Codon optimized synthetic cDNAs coding for human CLCF1 tagged with the protein C epitope (EDQVDPRLIDGK), human CRLF1-T2A-CLCF1 , mouse CLCF1, human CRLF1-SGGG linker-GPI anchor (CNTFR a.a 346-372) fusion protein, and human CNTFRα were generated by GeneArt (Thermo Fisher Scientific, Burlington ON).

Techniques: Staining, Flow Cytometry, Fluorescence, Isolation, Control