Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry of fresh whole-blood samples from anonymous donors. C4 protein was quantified (Median Fluorescent Intensity) in major immune cell types using flow cytometry. Jurkat cell line is the negative control and hepG2 is the positive control cell line (top 2 panels in dark gray). Rabbit IgG isotype controls for each major immune cell type are shown in the panel below the C4 protein quantification in that immune cell type (light gray). The bottom panel shows FMO control (dark gray). (b) Median Fluorescence Intensity (MFI) in major immune cell types in ten healthy donors from the Stanford Blood Center, as measured by flow cytometry. Group differences between immune cell types were tested using the nonparametric Mann-Whitney U Test and significance was tested using the false discovery rate (FDR). * = p <0.05, ** = p <0.01, *** = p <0.001. (c) Comparisons that survived threshold for multiple comparisons are shown. A comprehensive list of comparisons is provided in Supplementary Table 3. (f) Average of Median Fluorescence Intensity (MFI) of C4 protein signal in major immune cell types in ten healthy donors from the Stanford Blood Center. An isotype control from the rabbit IgG isotype control for each immune cell type was also provided.

Article Snippet: Then, cells were resuspended in permeabilization buffer and an antibody against the C4 protein beta chain with a conjugated fluorescent tag (Complement C4 polyclonal antibody, AbBy Fluor 647 Conjugated, Bioss, Woburn, MA) for 30 minutes at room temperature with rotation in the dark.

Techniques: Flow Cytometry, Negative Control, Positive Control, Control, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry was used to quantify C4 protein in major immune cell subtypes. (b) C4 protein was measured by immunoblotting against C4 protein (22233 Proteintech, Rosemont IL) using WES (capillary-based western blotting) in isolated neutrophils sampled from 15 SCZ and 21 control individuals. Samples were normalized using cellular actin (8H10D10, Invitrogen, Waltham, MA, USA). (c) Table showing the Descriptive statistics of measured immune cell-associated C4 protein in samples from individuals with SCZ compared to controls using different methods. Group comparisons of C4 protein for each major immune cell type (and plasma) were performed using one-way ANOVA. (d-f) CM and NCM were isolated from frozen live PBMC samples. Isolated cells were incubated, fixed, and stained for C4 protein (antibody directed against C4 protein, Proteintech, 22233-1-AP). C4 protein is localized throughout monocyte cells but is preferentially localized at the periphery. Representative images of CMs stained with Hoeschet stain for DNA and fluorescently labeled antibody against C4 protein in a sample from a control participant and a sample from a participant with SCZ. (e) Quantification of C4 protein throughout each cell was performed by measuring the Mean Intensity (Mean) from immunofluorescent images of C4 protein using Fiji. A mask was created from the nuclear stain and used to subtract the central C4 protein fluorescence to determine the Peripheral C4 protein Mean Fluorescence. (f) Table showing the exploratory descriptive statistics of CM and NCM C4 protein in samples from individuals with SCZ compared to controls. RFU = Relative Fluorescent Unit.

Article Snippet: Then, cells were resuspended in permeabilization buffer and an antibody against the C4 protein beta chain with a conjugated fluorescent tag (Complement C4 polyclonal antibody, AbBy Fluor 647 Conjugated, Bioss, Woburn, MA) for 30 minutes at room temperature with rotation in the dark.

Techniques: Flow Cytometry, Western Blot, Isolation, Control, Clinical Proteomics, Incubation, Staining, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry of fresh whole-blood samples from anonymous donors. C4 protein was quantified (Median Fluorescent Intensity) in major immune cell types using flow cytometry. Jurkat cell line is the negative control and hepG2 is the positive control cell line (top 2 panels in dark gray). Rabbit IgG isotype controls for each major immune cell type are shown in the panel below the C4 protein quantification in that immune cell type (light gray). The bottom panel shows FMO control (dark gray). (b) Median Fluorescence Intensity (MFI) in major immune cell types in ten healthy donors from the Stanford Blood Center, as measured by flow cytometry. Group differences between immune cell types were tested using the nonparametric Mann-Whitney U Test and significance was tested using the false discovery rate (FDR). * = p <0.05, ** = p <0.01, *** = p <0.001. (c) Comparisons that survived threshold for multiple comparisons are shown. A comprehensive list of comparisons is provided in Supplementary Table 3. (f) Average of Median Fluorescence Intensity (MFI) of C4 protein signal in major immune cell types in ten healthy donors from the Stanford Blood Center. An isotype control from the rabbit IgG isotype control for each immune cell type was also provided.

Article Snippet: Finally, cells were stained for C4 protein (bs-15186R-BF647, Bioss, Woburn, MA) after antibody optimization by incubation in antibody in Perm/wash buffer.

Techniques: Flow Cytometry, Negative Control, Positive Control, Control, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Isolated neutrophils from a healthy donor labeled for C4 protein (show in red, labeled with anti-C4 antibody (Proteintech 22233)) and DNA (shown in blue, stained with DAPI). The overlay shows that the C4 protein is at the rim of the nuclear DNA. (b) The Number of C4 gene copies for the various forms of the C4 gene (AS, AL, BS, and BL) was determined using ddPCR. Descriptive statistics for the control and SCZ groups in the Clinical Comparison Cohort are also provided. (c) Spearman correlation between measured Neutrophil C4 protein and the number of C4A gene copies is provided for the control, SCZ, and whole cohorts. The correlation between C4 protein associated with Neutrophils and the number of C4A gene copies was found to be statistically significant (r = 0.64, p = 0.01). The difference in correlations in individuals with SCZ and in controls trended towards significance based on the modified Fisher’s Z -transformation test for rho ( z [difference] = −1.68, p = 0.09). (d) Table showing the Spearman’s correlation of the number of C4A gene copies and the measured C4 protein associated with Neutrophils and CM in the SCZ, control, and whole (combined SCZ and control) cohorts.

Article Snippet: Finally, cells were stained for C4 protein (bs-15186R-BF647, Bioss, Woburn, MA) after antibody optimization by incubation in antibody in Perm/wash buffer.

Techniques: Isolation, Labeling, Staining, Control, Comparison, Modification, Transformation Assay

Journal: bioRxiv

Article Title: Peripheral Complement C4 Protein in Schizophrenia: Association with Gene Copy Number and Immune Cell Subtypes

doi: 10.1101/2025.09.16.676439

Figure Lengend Snippet: (a) Flow cytometry was used to quantify C4 protein in major immune cell subtypes. (b) C4 protein was measured by immunoblotting against C4 protein (22233 Proteintech, Rosemont IL) using WES (capillary-based western blotting) in isolated neutrophils sampled from 15 SCZ and 21 control individuals. Samples were normalized using cellular actin (8H10D10, Invitrogen, Waltham, MA, USA). (c) Table showing the Descriptive statistics of measured immune cell-associated C4 protein in samples from individuals with SCZ compared to controls using different methods. Group comparisons of C4 protein for each major immune cell type (and plasma) were performed using one-way ANOVA. (d-f) CM and NCM were isolated from frozen live PBMC samples. Isolated cells were incubated, fixed, and stained for C4 protein (antibody directed against C4 protein, Proteintech, 22233-1-AP). C4 protein is localized throughout monocyte cells but is preferentially localized at the periphery. Representative images of CMs stained with Hoeschet stain for DNA and fluorescently labeled antibody against C4 protein in a sample from a control participant and a sample from a participant with SCZ. (e) Quantification of C4 protein throughout each cell was performed by measuring the Mean Intensity (Mean) from immunofluorescent images of C4 protein using Fiji. A mask was created from the nuclear stain and used to subtract the central C4 protein fluorescence to determine the Peripheral C4 protein Mean Fluorescence. (f) Table showing the exploratory descriptive statistics of CM and NCM C4 protein in samples from individuals with SCZ compared to controls. RFU = Relative Fluorescent Unit.

Article Snippet: Finally, cells were stained for C4 protein (bs-15186R-BF647, Bioss, Woburn, MA) after antibody optimization by incubation in antibody in Perm/wash buffer.

Techniques: Flow Cytometry, Western Blot, Isolation, Control, Clinical Proteomics, Incubation, Staining, Labeling, Fluorescence