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

Journal: Neuro-Signals

Article Title: Targeting Voltage-Dependent Calcium Channels with Pregabalin Exerts a Direct Neuroprotective Effect in an Animal Model of Multiple Sclerosis.

doi: 10.1159/000495425

Figure Lengend Snippet: Fig. 3. Other EAE/MS-relevant cell types show no response to pregabalin treatment in vitro. A. Cacna2d1 and Cacna2d2 mRNA expression in murine splenocytes isolated from naïve C57BL/6J mice and stimulated with or without an ti-CD3 and anti-CD28 antibodies to induce T cell receptor stimulation for 48 hours in culture. Real-time reverse transcription-PCR was performed using 18S rRNA as en dogenous control. B. Murine sple nocytes were isolated and stained with efluor670, cultured for 4 days with pregabalin or without pregabalin (Ctrl) and thereafter the proliferation was assessed by flow cytometry C. Ca2+ imaging ex periments using Fura-2 in control and pregabalin treated splenocytes were performed (left) and quanti fied (right). Cross-linked anti-CD3 antibodies for T cell receptor stim ulation (CD3 X-link) were added as indicated by the arrow. D. (left): Cacna2d1 and Cacna2d2 expres sion in murine microglia after 24 hours’ stimulation with 100 U*ml-1 IFN-γ and without stimulation determined by rRT-PCR using 18S rRNA for normalization (right): Unstimu lated and inflamed microglia cultures were analyzed for the expression of MHC-II, CD86 and CD40 by flow cytometry with and without pregabalin. E. rRT-PCR analyses for Cacna2d1 and Cacna2d2 auxiliary subunits in primary mouse brain endothelial cells under naïve and inflammatory conditions. F. rRT-PCR analyses for the adhesion molecules Icam-1, Vcam-1 and the tight junction proteins Claudin-5 and Zo-1 and their expression in MBMECs under naïve and inflammatory conditions. G. TEER-measurement of the integrity of a monolayer of murine brain endothelial cells of naïve and inflamed cells with and without pregabalin over 24 h.

Article Snippet: The proliferation was assessed via flow cytometry (Beckman Coulter, Krefeld, Germany). rRT-PCR RNA was purified using TRIzol reagent (Life Technologies) and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was performed as previously described [33] by using TaqMan Gene Expression Assays (Life Technologies) with specific primers for mouse: Cacna2d1 (Mm00486607_m1), Cacna2d2 (Mm00457825_m1), Cacna1a (Mm00432190_m1), Cacna1b (Mm01333678_m1), Cacna1c (Mm01188822_m1), Cacna1d (Mm01209927_g1), Cacna1e (Mm00494444_m1), Cacna1f (Mm01352612_ m1), Cacna1s (Mm00489257_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), Claudin (Mm01320970_m1), Zo-1 (Mm00493699_m1) and eukaryotic 18S ribosomal RNA (18S rRNA) as endogenous control (VIC/MGB probe; 4319413E).

Techniques: In Vitro, Expressing, Isolation, Reverse Transcription, Control, Staining, Cell Culture, Flow Cytometry, Imaging, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Effect of Plasma Viremia on Apoptosis and Immunophenotype of Dendritic Cells Subsets in Acute SIVmac239 Infection of Chinese Rhesus Macaques

doi: 10.1371/journal.pone.0029036

Figure Lengend Snippet: Phenotypic analysis of CD1c + mDCs and pDCs subsets identified by two-color flow cytometry.

Article Snippet: In brief, the whole blood (100 μl) was incubated with the appropriate monoclonal antibodies against CD1c (clone AD5-8E7; Miltenyi), CD3ε (clone SP34; BD), CD4 (clone MT-466; Miltenyi), CD8 (clone BW/135/80; Miltenyi), CD11b (clone M1/70.15.11.5; Miltenyi), CD11c (clone 3.9; eBioscience), CD14 (clone M5E2; Biolegend), CD14 (clone TÜK4; Miltenyi), CD16 (clone VEP13; Miltenyi), CD20 (clone 2H7; Biolegend), CD20 (clone LT20; Miltenyi), CD40 (clone 5C3; BD), CD80 (clone L307.4; BD), CD83 (clone HB15e; BD), CD86 (clone FUN-1; BD), CD123 (clone 7G3; BD), CD209 (clone 120507; R&D), CCR5 (clone 3A9; BD), CCR7 (clone 150503; R&D), HLA-DR (clone G46-6; BD), and HLA-DR (clone L243; BD) for 15 min at room temperature.

Techniques: Cytometry, Migration