Bacteria were diluted in filtered saline, and fluorescent events were measured on a CyFlow Space cytometer (Sysmex Partec, France). Specifications of the apparatus and flow cytometric analyses are described in Text S1 .
Cyflow space cytometer
Manufactured by Sysmex
Sourced in Germany
The CyFlow Space cytometer is a compact and versatile flow cytometry system designed for a wide range of applications. It utilizes advanced optical and fluidic technologies to deliver accurate and reliable data for various cell analysis and sorting tasks.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green dye, by Lonza (1 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Flowmax software, by Sysmex (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fcs express 4, by De Novo Software (1 mentions)
Anti cd25 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd62l apc, by Thermo Fisher Scientific (1 mentions)
Anti cd3ε fitc, by Thermo Fisher Scientific (1 mentions)
8 protocols using cyflow space cytometer
1
Fluorescent Bacteria Analysis by Flow Cytometry
2
Efficient HDR Assessment Using Flow Cytometry and TIDER
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HDR efficiency was assessed as the percentage of either GFP-positive cells or edited alleles. Flow cytometry of the GFP+ cells was performed using a CyFlow Space cytometer (Sysmex-Partec, Germany). To measure the percentage of edited alleles, we sequenced the locus of interest and analyzed chromatograms from Sanger sequencing with “Tracking of Insertion, DEletions and Recombination events” (TIDER, http://shinyapps.datacurators.nl/tider/ , accessed on 31 March 2023) [23 (link),24 (link)]. Briefly, the program decomposes chromatogram peaks to detect overlapping subsequences and presents results as percentages of different alleles. The program uses a chromatogram of a mutated locus (non-edited) as a control sample and a chromatogram of a fully corrected locus (e.g., wild type eGFP) as a reference sample. This allows us to distinguish between HDR-corrected and NHEJ-corrected alleles and to evaluate their efficiencies separately.
3
Pollen Ploidy Analysis via Flow Cytometry
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Pollen ploidy in jas-3 mutants was analyzed by collecting open flowers from individual plants into eppendorf tubes, vortexing in 2mL of 100mM sodium phosphate buffer (pH 7) for 3min, and filtering through a 50µm nylon mesh. Pollen population is characterized by an elevated high angle scatter (SSC) and autofluorescence, which allows discrimination of haploid (1n) and diploid (2n) pollen, as previously described (Storme and Geelen, 2011; Erilova et al., 2009) . These two populations were gated and quantified (Supplemental Figure 2). For ploidy analysis of nuclei, leaf tissue was chopped in 2mL of Galbraith buffer (45 mM MgCl2, 20 mM MOPS, 30 mM sodium citrate, 1 % (v/v) Triton X-100. Adjust pH to 7.0) using a razor blade, filtered through a 50µm mesh, stained with SYBR Green dye (Lonza), and analyzed on a CyFlow Space cytometer (Sysmex).
4
Characterization of hSC Lines
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Flow cytometry was performed to characterize the cultured hSC lines for the presence of Pax7 protein, using anti-Pax7 antibody (Sigma Aldrich, St. Louis, MO, USA, SAB1412356) dilution 1:10, and the presence of CaSR protein using anti-CaSR (5C10, ADD) antibody (Thermo Fisher Scientific, Waltham, MA, USA, MA1-934) at two different dilutions of 1:500 and 1:100, both followed by goat anti-mouse IgG (H + L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, Waltham, MA, USA, A28175, dilution 1:200). The FIX & PERM® Cell Permeabilization Kit (Invitrogen, Walthan, MA, USA, GAS004) was used for fixing and permeabilizing cells in suspension. Then, 1 × 105 cells were labeled with primary antibodies for 20 min at room temperature (RT) in PBS with 1% bovine serum albumin (BSA), then removed and washed two times and centrifuged for 5 min at 1500 rpm; secondary antibody was incubated for 30 min at RT in the dark, then washed once and promptly analyzed. The stained cells were analyzed in a CyFlow®Space cytometer, equipped with FlowMax® software (Sysmex Partec, Norderstedt, Germany).
5
Immunophenotyping of hSkMC Cells
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hSkMC lines were evaluated by flow cytometry with a CyFlow®Space cytometer (Sysmex Partec), equipped with FlowMax® software. The antibodies used (Abcam) were directed against the following antigens (the tags are given in parentheses): CD44 (PE/Cy7), CD90 (APC), CD105 (FITC), CD45 (PerPC), CD34 (PE) and CD56 (PerCP Cy5.5) and PAX-7 (FITC). Each antibody was diluted according to manufacturer's instruction. Briefly, 1 × 105 cells were labeled with antibodies in PBS with 1% bovine serum albumin (BSA) for 20 min RT in the dark, then washed once and promptly analyzed.
6
Parasite Staining and Analysis
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After electrical stimulation, parasites were stained with 2 μg/ml Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) prior to fixation with 4% paraformaldehyde, or incubated further at 37°C for 5 or 24 h before staining. Samples were stored at 4°C until analysis. A CyFlow Space cytometer (Sysmex Partec, Görlitz, Germany) was used for analysis by exciting the samples through the UV laser. The data was analyzed with the FCS Express 4 (De Novo Software, Glendale, CA, USA).
7
Activation and Migration of T Cells
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TCL, loaded or not with NBR, were stimulated with MOG35–55 (10 μg mL−1) and analyzed after 12 and 24 hours in order to evaluate the expression of CD25 (activation) and CD62L (a key regulator of migration, early-modulated during cell activation). Cells were stained (20 minutes RT in the dark) with the following fluorescent anti-mouse monoclonal antibodies (all from eBioscience): anti-CD25 PE (1 : 100 diluted), anti-CD62L APC (1 : 600 diluted), anti-CD3ε FITC (1 : 100 diluted). Then the cells were analysed with a CyFlow Space cytometer (Sysmex-Partec, Germany).
8
NBR-loaded TCL Viability Evaluation
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NBR-loaded TCL were analysed for cell viability immediately after loading and upon in vitro stimulation for 24 and 72 hours with MOG35–55 10 μg mL−1 in complete medium. Cell viability was assessed by flow cytometry using the Annexin V Apoptosis Detection Kit FITC (eBioscence, U.S.A.) and the CyFlow Space cytometer (Sysmex-Partec, Germany).
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