Journal: EBioMedicine

Article Title: Rapid detection of bacterial infection using a novel single-tube, four-colour flow cytometric method: Comparison with PCT and CRP

doi: 10.1016/j.ebiom.2021.103724

Figure Lengend Snippet: A schematic diagram on how to calculate the FCBI-index value of the patient with microbiologically confirmed Streptococcus pneumoniae pneumonia using the novel FCBI-index method. (a) The incubation of the whole blood sample with the receptor-specific antibodies, red blood cell lysis, and flow cytometry run were carried out one after another in the same test tube. (b) During the flow cytometry run, the leukocyte population within gate R1 was first gated out from the side scatter (SSC)/MHCI bivariate histogram, after which gates R2 (red) and R3 (blue) were set around neutrophil and lymphocyte populations, respectively, in the SSC/CD64 bivariate histogram containing leukocytes inside gate R1. The stop count of 1000 monocytes inside the monocyte gate (black/R4) was used, leading to the collection of a total of 5000–20000 leukocytes. (c) For the purpose of data analysis, three additional bivariate histograms, namely SSC/CD35, SSC/CD64, and SSC/CD329 were made, showing mean fluorescence intensities (MFI, which correlate with the number of receptors on the cell surface) of gated neutrophil (red) and lymphocyte (blue) populations. In the SSC/CD35 histogram, B-lymphocytes expressing CD35 were left outside the lymphocyte gate. (d) The actual FCBI-index value was calculated by substituting the obtained receptor-specific MFI data from neutrophil and monocyte populations into the bacterial infection algorithm. A more detailed step-by-step description of the development of the FCBI-index method is presented in the method section and Supplementary Figs. 1–5. 1.54 × gCMF-HBSS: 1.54-fold calcium- and magnesium-free HBSS -buffer supplemented with 1.54 mg/mL gelatin.

Article Snippet: Fluorescence-conjugated receptor-specific monoclonal antibodies (test kit): Phycoerythrin (PE)-conjugated anti-human CD35 (Clone E11) was purchased from Ancell (ANC-184-050), PerCP-Vio700-conjugated anti-human CD64 (Clone 10.1.1) was purchased from Miltenyi Biotec (130-101-422), fluorescein isothiocyanate (FITC)-conjugated anti-human CD329 (Clone 191240) was purchased from R&D systems (FAB1139F) and AF647-conjugated anti-human MHC class I molecule (Clone W6/32) was purchased from R&D systems (FAB7098R). (Supplementary Table 5).

Techniques: Incubation, Red Blood Cell Lysis, Flow Cytometry, Fluorescence, Expressing, Infection

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet: Flow cytometry enriched antibody cocktail (22 μL) panel for analyzing rarer granulocyte and myeloid cells in mouse spleen for downstream scRNA-seq

Article Snippet: FcεRIα Antibody, anti-mouse, PE-Vio® 615, REAfinityTM Clone REA1079 , Miltenyi Biotec , Cat# 130-118-899; RRID: AB_2801723.

Techniques: Flow Cytometry, Marker

Journal: STAR Protocols

Article Title: Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics

doi: 10.1016/j.xpro.2022.101402

Figure Lengend Snippet:

Article Snippet: FcεRIα Antibody, anti-mouse, PE-Vio® 615, REAfinityTM Clone REA1079 , Miltenyi Biotec , Cat# 130-118-899; RRID: AB_2801723.

Techniques: Recombinant, Sterility, Saline, Extraction, Blocking Assay, Software, Solvent, Sequencing, RNA Sequencing, Transferring, Aerosol, Irradiation, Inverted Microscopy, Flow Cytometry, Fluorescence