Splenocytes and lymphocytes were obtained by squeezing the spleen and the lymph nodes. Ammonium-Chloride-Potassium lysing buffer was used to remove red blood cells. Single cells on the grafted lung tissue were obtained using a gentleMACS™ Octo Dissociator and the Lung Dissociation Mouse Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of CD19-positive cells among lymphocytes defined the B-cell population. Single cells were stained with the following fluorochrome-labeled anti-mouse antibodies: anti-CD3-APC/Cy7 (clone 17A2, BioLegend, San Diego, CA, USA) and anti-CD19-PE (clone 6D5, BioLegend, San Diego, CA, USA). Cells were acquired on the BD LSRFortessa II analyzer (BD Biosciences, San Jose, CA, USA). The mean fluorescence intensity (MFI) was assessed using FlowJo software (BD Life Sciences, Franklin Lakes, NJ, USA).
Lsrfortessa 2 analyzer
Manufactured by BD
Sourced in United States
The BD LSRFortessa II analyzer is a flow cytometry instrument designed for advanced multiparametric analysis of biological samples. It provides high-performance detection and data acquisition capabilities for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Collagenase 4, by Merck Group (2 mentions)
Zombie aqua fixable viability kit cell dye, by BioLegend (2 mentions)
Intracellular staining kit for foxp3 and ifn γ, by BioLegend (2 mentions)
α cd16 32 ab clone 93, by Thermo Fisher Scientific (2 mentions)
Anti cd19 pe clone 6d5, by BioLegend (1 mentions)
Gentlemacs octo dissociator, by Miltenyi Biotec (1 mentions)
Ab97229, by Abcam (1 mentions)
Accuri c6, by BD (1 mentions)
Cytofix, by BD (1 mentions)
Cytoflex lx, by Beckman Coulter (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Intrastain kit, by Agilent Technologies (1 mentions)
7 protocols using lsrfortessa 2 analyzer
1
Isolation and Characterization of Lung Immune Cells
2
Dissociation and Staining of Tumor Tissues
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Collected tumor tissues were minced and incubated in RPMI 1640 medium containing 2% FBS, 1mg/mL collagenase IV (Sigma: #C5138), 0.1mg hyaluronidase (Sigma: #H6254), and 200U DNase I (Sigma: #D5025) at 37°C for 1 hours to make single cells. In vitro virus-infected cells or single cells from tumor tissues were blocked with α-CD16/32 Ab (clone 93, eBioscience: #14-0161-85; 1:1000) and then cells were stained with 100 μL Zombie Aqua Fixable Viability Kit cell dye (BioLegend, San Diego, CA, USA) at a dilution of 1:1000 and left in room temperature for 15 min in the dark. Cells were stained in 100 μL total stain volume (50 μL BV stain buffer, 50 μl 2% FBS) with antibody at a dilution of 1:200 for 30 min on ice in the dark. The sources of antibodies are listed in Supplementary Table 1 . The intracellular staining kit for Foxp3 and IFN-γ was purchased from BioLegend. Cells were then washed once with FACS buffer and fixed in 1% paraformaldehyde (EK Industries, Joliet, IL, USA) before being stored at 4°C overnight and acquired the next day on a BD LSR Fortessa II analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed using flowJo cytometer software.
3
Detecting Donor-Specific Antibodies by Flow Cytometry
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Donor-specific antibodies (DSAs) in recipient blood were detected by flow cytometric cross match as previously described [29 (link)]. Briefly, splenocytes obtained from the donor’s spleen were prepared and incubated in recipient serum samples for 30 min. After washing, the cells were incubated with goat anti-mouse IgG (ab6785; Abcam, Cambridge, UK) and IgM (ab97229; Abcam, Cambridge, UK), along with anti-mouse CD3-APC/Cy7 antibodies (clone 17A2, BioLegend, San Diego, CA, USA), for T-cell gating by flow cytometry. Subsequently, the cells and the antibodies were analyzed using the BD LSRFortessa II analyzer (BD Biosciences, San Jose, CA, USA). MFI was assessed using the FlowJo software.
4
TNFSF Ligand Expression Profiling
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Cells were washed and resuspended in FACS buffer (PBS + 0.2% w/v BSA + 0.02% w/v NaN3). For TNFSF ligands, cells were labeled with 20 µg/ml of each antibody or matching isotype control. Staining was performed at room temperature for 30 min in the dark, after which cells were washed twice in FACS buffer and resuspended in BD Cytofix (BD Biosciences). Antibodies against lineage markers were used per manufacturers’ instructions. Flow cytometry data was acquired using the BD LSRFortessa II analyzer, BD Accuri™ C6 (BD Biosciences) and CytoFlex LX (Beckman Coulter; Brea, CA). For TNFSF ligand evaluation, 10,000 CD19+CD3- B cells were acquired. Fluorescence minus five (FM5) controls labeled with CD3 AF700 and CD19 FITC, as well as control IgG antibodies for AF647, AF405, PE, BUV395 and BV711 antibodies, were used to set upper limits for background signals on the missing labels. Data were analyzed using FlowJo v10 (FlowJo, LLC; Ashland, OR).
5
Dissociation and Staining of Tumor Tissues
6
Cardiac Differentiation of Mouse ES Cells
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Mouse ES cells on day 14 of cardiac differentiation were analyzed with the use of an LSR Fortessa II Analyzer (BD Biosciences) and FACSDiva 7.0 software as previously described [33 (link)]. In brief, the cultured cells were isolated by exposure to 0.25% trypsin/EDTA (Sigma-Aldrich) for 6 min at 37°C under 5% CO2. These were then fixed and permeabilized with IntraStain Reagent A and B of an IntraStain kit (DAKO) according to the manufacturer’s protocol. Primary antibodies included antibodies to TNNT2 (1/200 dilution, goat polyclonal, HyTest) and to GFP (1/500 dilution, rabbit polyclonal, Molecular Probes). Secondary antibodies included Alexa Fluor 647–conjugated donkey antibodies to goat immunoglobulin G and Alexa Fluor 488–conjugated donkey antibodies to rabbit immunoglobulin G (Molecular Probes). To assay apoptosis, Annexin V positive apoptotic cells were measured using a Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide Kit (Molecular Probes) according to the manufacturer’s protocol.
7
Quantifying Reactivation and Expression in T-Cell Models
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GFP fluorescence, reactivation percentage, and CXCR4 expression were quantified using a BD LSR Fortessa II analyzer. Based on forward and side scatter, gates were set up to exclude debris and filter out single events that consist of two independent particles. 30,000 cells in total were collected. All flow cytometry data were analyzed using FCS Express 6 (Figure S2 ). A gate for JLat or TLat reactivation in the side scatter vs. fluorescein isothiocyanate (FITC) scatter plot was created using the untreated measurement similar to previous reports [6 (link),20 (link)].
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