Cells were cultured in DMEM-GlutaMax supplemented with 10% FCS (unless otherwise specified), 100 U/mL penicillin and 100 µg/mL streptomycin, in the continuous presence of geneticin, in a water-saturated atmosphere containing 7.5% CO2. To study cellular proliferation, equal numbers of cells (3 × 104) were seeded on 12-well plates in DMEM-GlutaMax supplemented with either 10% FCS, 1% FCS, or 1% FCS + 100 nM NDP-MSH. Cells were allowed to attach and recover for 24 h, then manually counted using a hemocytometer at different times from 24 to 96 h. Doubling times were calculated by nonlinear regression using an exponential growth equation and GraphPad Prism Software (https://www.graphpad.com , San Diego, CA, USA). For cell cycle analysis, cells fixed in ethanol 70% in PBS were pelleted, resuspended in PBS, and further treated with 100 µg/mL RNase A and 40 µg/mL propidium iodide (PI). At least 104 cells were analyzed in a LSRFortessa X-20 cytometer (BD Biosciences, Franklin Lakes, NJ, USA), using ModFit LTTM software (https://www.vsh.com/products/mflt/ ).
Lsrfortessa x 20 cytometer
Manufactured by BD
Sourced in United States
The BD LSRFortessa X-20 is a flow cytometer designed for multiparameter analysis. It features 20 independent detectors and supports a wide range of fluorescent dyes. The instrument is capable of high-speed data acquisition and can be configured with various laser and detection options to meet specific research needs.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (3 mentions)
Facsdiva software, by BD (3 mentions)
Diva software, by BD (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Rnase a, by Merck Group (2 mentions)
Facsdiva v6, by BD (2 mentions)
Fixable aqua dead cell stain, by Thermo Fisher Scientific (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Human fc block, by BD (2 mentions)
Perm wash buffer, by BD (2 mentions)
Anti cd16 32 clone 93, by Thermo Fisher Scientific (2 mentions)
Pe annexin 5 apoptosis detection kit, by BD (2 mentions)
Cell viability dye zombie uv, by BioLegend (2 mentions)
Cytoperm buffer, by BioLegend (2 mentions)
Compbead, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Facsaria fusion, by BD (2 mentions)
73 protocols using lsrfortessa x 20 cytometer
1
Cell Proliferation Assay with NDP-MSH
2
Comprehensive Immune Cell Profiling
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Total blood cells or leukocytes from the liver or spleen were incubated with Fc block (eBiociences, Thermo Fisher Scientific) for 10 minutes at 4-8°C. Surface staining was performed for 20 minutes at 4-8°C and always included Fixable Viability Dye (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) for exclusion of dead cells. The following conjugated anti-mouse mAbs (and respective clones) were used: CD3 PerCP-Cy5.5 (145-2C11), CD4 APC or Brilliant Violet (BV) 510 (GK1.5), CD8 BV711 (53-6.7), CD62L FITC (MEL-14), CD19 PE (1D3), CD25 APC (3C7), CD44 PE-Cy7 (IM7), CD45 Alexa Fluor 700 (30-F11), CD69 BV650 (H1.2F3), CD127 PE-Dazzle 594 (A7R34), NK1.1 PE-Cy5 (PK136), KLRG1 PE (MAFA), CXCR3 APC (CXCR3-173), TCR γδ BV421 (GL3), all from either BioLegend (San Diego, CA, USA) or Sysmex (Kōbe, Hyōgo, Japan). Cells were acquired in a BD LSR Fortessa X-20 cytometer and analyses were performed within live, single (based on FSC-A vs. FSC-W parameters) CD45+ leukocytes using FlowJo v10 (FlowJo, BD). For the clustering and visualization of high-dimensional data, equivalent numbers of live, single CD45+ cells from each condition were concatenated. Clustering was performed using X-shift (number of clusters determined by the algorithm) and data are presented using the dimensionality reduction method TriMAP (large-scale dimensionality reduction using triplets).
3
Th17 Cell Identification Protocol
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In vitro or isolated primary cells were stimulated with 20 ng/ml PMA, 1 μg/ml ionomycin, and 10 mg/ml brefeldin A (GolgiStop, BD Biosciences) for 2 h at 37°C. Cells were stained with fixable viability stain (Zombie Aqua, BioLegend). Cells were stained for surface markers followed by intracellular cytokine staining using listed antibodies in Table S1 with eBioscience Foxp3/Transcription Factor Staining Buffer. Analysis by flow cytometry was conducted using a BD LSRFortessa X-20 cytometer and data were analyzed using FlowJo (v10.8.1). Th17 cells were defined and analyzed by gating on live, CD45+, CD3ε+, CD4+, RORγt+ cells.
4
Multiparameter Immunophenotyping of Lymphoid Cells
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Immunophenotyping was performed on thymic and splenic lymphoid populations by five-color fluorescence analysis, according to standard protocols. The following anti-mouse antibodies were used: CD8-PE (catalog number 553041), IgM-APC (catalog number 550676), CD4-PE.Cy7 (catalog number 552775), CD25 PerCPCy5.5 (catalog number 551071) from BD Biosciences, Franklin Lakes, NJ, USA; B220-PercPCy5.5 (catalog number 1116180) and CD44 BV510 (catalog number 1115215) from Sony Biotechnology Inc., San Jose, CA, USA. Cells were analyzed on a LSR FORTESSA X-20 cytometer (BD Biosciences) immediately after incubation with Molecular Probes Sytox Blue Dead Cell Stain (catalog number S34857) (Life Technologies) to exclude dead cells.
5
Multiparametric Flow Cytometry Analysis
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Samples were incubated with a Live Dead Aqua Blue reagent (Thermo Fisher Scientific) according to manufacturer instructions. After one wash, cells were saturated with 1% BSA-FcR Blocking (Miltenyi) in DPBS for 15 min at 4°C, and then surface-stained for 20 min at 4°C with a cocktail of coupled-antibodies (S2A Table ). Leukocytes were then fixed for 20 min at room temperature with 4% paraformaldehyde, permeabilized with 0.05% Saponine-DPBS, and stained with coupled-antibodies directed against intracellular cytokines (S2B Table ). Samples were finally analyzed with a LSR Fortessa X-20 cytometer (BD Bioscience). Fluorochrome compensation was performed with compensation beads (BD Bioscience) and FMO (Fluorescence Minus One) conditions.
6
Flow Cytometry for Cell Sorting and Analysis
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Cell sorting was performed on a FACSAria W or L (BD Biosciences) cell sorter. For IL-2Rα and CD28 level sorting, cells were labelled with anti-CD28-PECy7 (clone 37.51, eBioscience) or anti-CD25-FITC (clone 7D4, BD).
Flow cytometry was performed on a FACSCanto II or LSRFortessa X-20 cytometer (both BD Biosciences). Data were analysed using FlowJo software (Treestar).
A known number of beads (Rainbow calibration particles, BD Biosciences) and propidium iodide (0.2 μg ml−1, Sigma) was added to samples immediately prior to analysis. The ratio of beads to live cells was used to estimate the absolute cell number. The following monoclonal antibodies were used for the detection of cell surface markers: anti-CD25 -PECy7, or—APC (clone PC61, BD Biosciences) anti-CD28-PECy7 (clone 37.51, eBioscience). Staining was performed in PBS containing 0.1% BSA and 0.1% sodium azide (Sigma). InSupplementary Fig. 8 , activated cells were defined as the 50% of cells with the highest FSC fluorescence. Spearman's correlation was calculated using Matlab 2011a's corr function.
Flow cytometry was performed on a FACSCanto II or LSRFortessa X-20 cytometer (both BD Biosciences). Data were analysed using FlowJo software (Treestar).
A known number of beads (Rainbow calibration particles, BD Biosciences) and propidium iodide (0.2 μg ml−1, Sigma) was added to samples immediately prior to analysis. The ratio of beads to live cells was used to estimate the absolute cell number. The following monoclonal antibodies were used for the detection of cell surface markers: anti-CD25 -PECy7, or—APC (clone PC61, BD Biosciences) anti-CD28-PECy7 (clone 37.51, eBioscience). Staining was performed in PBS containing 0.1% BSA and 0.1% sodium azide (Sigma). In
7
Cell Proliferation Assays for Diverse Applications
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For the resazurin proliferation assay, cells were seeded at a density of 150,000 cells/ml in 96-well plates. At 24 and 48 h, 10 µl of CellTiter-Blue (Promega) was added, and cells were further incubated at 37°C for up to 4 h. Absorbance at 560/595 nm was measured and analysed according to the manufacturer’s instructions using a Tecan Infinite 200 Pro plate reader (Tecan Life Sciences). For CSFE incorporation–based proliferation assay, cells were incubated overnight with 3 mM of hydroxyurea (Sigma-Aldrich), washed, and labelled with CFSE (Invitrogen) according to the manufacturer’s instructions, resuspended in complete medium and cultured for 4 d. Cells were then analysed using an LSRFortessa X-20 cytometer and FlowJo software (BD Biosciences). Proliferation of HEK cells was analysed as a percentage of confluency. For that, cells were seeded in complete culture medium at a density of 6,250 cells/cm2 for 24 h and placed in an IncuCyte Zoom Live-Cell analysis system (Sartorius). Phase contrast and GFP fluorescence pictures were taken every 3 h and analysed using a confluency mask.
8
Adoptive Transfer of Infected Alveolar Macrophages
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Mouse AMs were isolated from WT, Mavs-/- or ICAM-1-/- mice (Female, 8 to 10 weeks old) as described above, and then infected with GenRM.avium strains at an MOI of 10 for 24 hr at 37°C and 5% CO2. Infected AMs were washed with pre-cold PBS thrice and then intratracheally injected into mice at a dose of 2x105 cells/mouse in 15 μl PBS. At the time of AM injection, recipient mice had been infected with GenSM.avium for 3 weeks as described [32 (link)]. At day 1, 3 and 6 post injection, mouse lungs were harvested, homogenized, and spread on 7H10 plus 10% OADC with or without gentamycin (200μg/ml). Bacterial number was counted 10–15 days after incubation at 37°C. For AM survival assay in mice after adoptive transfer, GenRM.avium-infected AMs were labeled with Vybrant® CFDA SE Cell Tracer Kit before intratracheal injection. Single mouse lung cells were prepared as described previously at day 1, 3 and 6 post injection34, and analyzed by BD LSRFortessa X-20 cytometer.
9
PBMC Flow Cytometry Activation Panel
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Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples and used for the quantification of cellular activation by flow cytometry. PBMCs were stained with antibodies: BUV BUV395-anti-CD45 (BD Horizon), BV605-anti-CD3 (Biolegend), ECD-anti-CD4 (Beckman Coulter), APC-H7-anti-CD8 (BD Pharmingen), AF700-anti-CD38 (BD Pharmingen), BV785-anti-HLA-DR (Biolegend), as well as Aqua intracellular dye for dead cells (Invitrogen; L34957). Specimens were analyzed using a LSRFortessa X-20 cytometer (BD Biosciences), and inter-experiment consistency was calibrated using rainbow bead (Spherotech URFP-30-2). Proportional quantification of cellular activation subsets was done using FlowJo (Treestar, Woodburn, Oregon, USA).
10
CRISPRi-Mediated Transcriptional Regulation Assay
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The CRISPRi-KAT5-sg1/sg2, CRISPRi-KAT8, or parental Jurkat 2D10 cells were first treated with the various LRAs or MG-149 (APExBIO) at the indicated concentrations for 20 hr, and then subjected to flow cytometry on a BD Bioscience LSR Fortessa X20 cytometer for GFP fluorescence. The data were analyzed using Flowjo software. Each drug treatment was done in 200 μl RMPI medium (Invitrogen) with 10% FBS (Gemini 900108 Lot A96C). To induce CRISPRi-mediated downregulation of KAT5 or KAT8, the CRISPRi-KAT5-sg1/sg2, CRISPRi-KAT8 cells were pre-treated for 48 hr with 1 μg/ml Dox before the LRA treatments. For control groups, 0.1% DMSO was used.
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