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: Surface-labelled cells were fixed and permeabilized using the FoxP3/Transcription factor staining buffer set, incubated with 2% normal rat serum (STEMCELL Technologies, Vancouver BC) at room temperature for 15 min, and stained with Alexa Fluor 647-conjugated anti-CLCF1 mAb (1 μg/ml) at room temperature for 45 min. Fluorescence was quantified using a FACSymphony A1 flow cytometer.

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

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: Surface-labelled cells were fixed and permeabilized using the FoxP3/Transcription factor staining buffer set, incubated with 2% normal rat serum (STEMCELL Technologies, Vancouver BC) at room temperature for 15 min, and stained with Alexa Fluor 647-conjugated anti-CLCF1 mAb (1 μg/ml) at room temperature for 45 min. Fluorescence was quantified using a FACSymphony A1 flow cytometer.

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

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: Surface-labelled cells were fixed and permeabilized using the FoxP3/Transcription factor staining buffer set, incubated with 2% normal rat serum (STEMCELL Technologies, Vancouver BC) at room temperature for 15 min, and stained with Alexa Fluor 647-conjugated anti-CLCF1 mAb (1 μg/ml) at room temperature for 45 min. Fluorescence was quantified using a FACSymphony A1 flow cytometer.

Techniques: Staining, Flow Cytometry, Fluorescence, Isolation