HA‐Dox was adsorbed onto murine RBC at RT in 55% serum. Washed naïve RBC and washed RBC:HA‐Dox suspensions (5 μl) at 10% Hematocrit were added to 995 μl of PBS, gently vortexed, and ran on a BD LSRFortessa cell analyzer (BD Biosciences, San Jose, CA), gated at 10,000 events. Data were analyzed using FCS Express Version 6 (DeNovo Software, Pasadena, CA). Washed RBC and HA‐Dox suspensions at 10% Hemocrit were incubated at room temperature with annexin V‐647 in buffer containing 2 mM CaCl2 for 15 min. After incubation, an aliquot was aspirated into a BD LSRFortessa cell analyzer (BD Biosciences) for analysis, gated at 10,000 events. Results were expressed as percentage of Annexin V positive RBCs. Data were analyzed using FCS Express Version 6 (DeNovo Software). Homogenates (100 μl) from intestinal organs (duodenum, jejunum, ileum, cecum, colon), diluted in PBS (900 μl) were gently vortexed, and directly aspirated into a BD LSRFortessa cell analyzer (BD Biosciences) for analysis, gated at 10,000 events.
Lsrfortessa cell analyzer
Manufactured by BD
Sourced in United States, Germany, United Kingdom, Canada, Belgium, Switzerland, China, Japan
The BD LSRFortessa cell analyzer is a flow cytometry instrument designed for high-performance cell analysis. It features multiple laser excitation sources and detectors to enable the simultaneous measurement of multiple cellular parameters. The core function of the BD LSRFortessa is to provide researchers with a versatile and efficient tool for analyzing heterogeneous cell populations.
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Lab products found in correlation
Facsdiva software, by BD (53 mentions)
Ionomycin, by Merck Group (12 mentions)
Facsdiva, by BD (12 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (11 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (11 mentions)
Facsaria 3 cell sorter, by BD (10 mentions)
Cytofix cytoperm kit, by BD (10 mentions)
Trypsin edta, by Thermo Fisher Scientific (10 mentions)
Golgiplug, by BD (9 mentions)
Facsaria 2 cell sorter, by BD (9 mentions)
Propidium iodide, by Merck Group (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
Propidium iodide, by Thermo Fisher Scientific (9 mentions)
Mitotracker green, by Thermo Fisher Scientific (9 mentions)
Cytofix cytoperm, by BD (8 mentions)
Fc block, by BD (8 mentions)
Facsaria 2, by BD (8 mentions)
Zombie aqua fixable viability kit, by BioLegend (7 mentions)
Golgistop, by BD (7 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (7 mentions)
1 144 protocols using lsrfortessa cell analyzer
1
Quantifying Erythrocyte Binding and Annexin V Positivity
2
Macrophage Uptake Inhibition Assay
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PMs were seeded in 6-well plates at 3 × 106 cells per well. For inhibition experiments, macrophages were preincubated with 50 μM amiloride, 12.5 μM chlorpromazine, and 7.5 mM MβCD for 30 min and then incubated with 10 μM IR-61 for another 30 min. Cells were then washed and treated with 1 mM EDTA for 5 min, gently harvested, and transferred to microcentrifuge tubes on ice. After washing for 3 times and resuspension in flow cytometry buffer, at least 10,000 cells per sample were analyzed using a BD LSRFortessa cell analyzer (BD Biosciences). For fluorescence microscopy analysis, the nuclei of PMs were stained using Hoechst33258 after treating as described above. Fluorescent images were captured by an NIR fluorescent microscope (Leica, excitation/emission: 770/830). The mean fluorescent intensity was assessed using the Leica LAS AF Lite software. For flow cytometric analysis of IR-61 uptake in macrophages with different activation state, PMs were seeded in 6-well plates (3 × 106 cells per well) and then added 100 ng ml−1 LPS (#L8880, Solarbio) or 20 ng ml−1 IL-4 (#96-214-14-5, Peprotech), respectively, for 24 h to induce M1 or M2 polarization. IR-61 was added and incubated with cells for another 2 h, then macrophages were harvested and analyzed using a BD LSRFortessaTM cell analyzer (BD Biosciences).
3
Cytotoxic NK Cell Activation Assay
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Healthy human donor PBMNCs or monkey PBMNCs were co-cultured without or with K562 cells (ATCC) in RPMI1640 medium with 10% FBS and 1x pen-strep antibiotics. Effector to target cells ratios of 5:1 or 10:1 were tested. After two hours target cell stimulation, PBMNCs were stained with Live/Dead viability dye, antibodies to CD3, CD14, CD20, NKG2A, CD16 and CD56, and the degranulation marker CD107a for flow cytometric analysis on the BD LSRFortessa™ Cell Analyzer. For cytokine analyses, PBMNCs were treated with IL-12 (10ng/ml), IL-15 (10ng/ml) and IL-18 (100ng/ml) overnight or stimulated with K562 at effector: target ratio at 5:1 or 10:1 for 1 hour, then monensin (Golgi-Stop, BD Biosciences) and brefeldin A (Golgi-Plug, BD Biosciences) were added per product instructions. 4-5 hours later, cells were washed and stained with Live/Dead viability dye, antibodies to CD3, CD14, CD20, NKG2A, CD16 and CD56. Cells were washed and then fixed with Cytofix/Cytoperm (BD Biosciences), followed with intracellular staining with anti-IFNγ and anti-TNFα mAb in PermWash buffer (BD Biosciences). Multi-parameter flow cytometry analysis was performed on BD LSRFortessa™ Cell Analyzer.
4
Multiparametric Flow Cytometry Analysis
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Cells from in vitro cultures or single cell suspensions obtained from organs from euthanized mice were stained with mAbs: anti-mouse CD4, anti-mouse CD8, anti-mouse CD3 and anti-mouse CD90 (BD Biosciences Pharmingen, San Diego, CA). Propidium iodide was used to evaluate viability and to eliminate dead cells from phenotypic analyses. Mean fluorescence intensity in the forward scatter channel (FSC) was used to estimate relative cell size. Cell cycle analysis was performed as described [73 (link)]. Cells were analyzed on a BD LSRFortessa Cell Analyzer (BD Biosciences) and the data analyzed with FlowJo software (Tree Star, Inc., San Carlos, CA). EGFP and EYFP signals were separated with a 526 nm dichroic mirror and 510/20 nm and 550/30 nm bandpass filters. Where indicated, numbers of viable cells were determined by cell counting using a BD LSRFortessa Cell Analyzer connected to a BD High Throughput Sampler (BD Biosciences). The number of live cells in 100 μl of cell suspension was determined in triplicates.
5
Apoptosis and ROS Analysis in HPDE6-C7 Cells
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The percentage of apoptotic cells was determined using an annexin V-FITC/PI apoptosis detection kit. HPDE6-C7 cells were incubated in 6-well plates in different culture media simultaneously. First, HPDE6-C7 cells were digested with 0.25% trypsin, collected in phosphate buffered saline (PBS) and pelleted by centrifugation at 1000 rpm for 5 minutes. Then, the cells collected in binding buffer were stained with 5 μL annexin V-FITC and 5 μL propidium iodide (PI). Finally, the cells were cultured at room temperature away from light for 15 minutes and analyzed by flow cytometry (BD LSRFortessa Cell Analyzer, BD Biosciences, Franklin Lakes, NJ).
Reactive oxygen species generation was assessed using a ROS Detection Assay Kit following the manufacturer's instructions. HPDE6-C7 cells from different groups were collected, and the cell pellets were resuspended in culture medium containing 1 × ROS label. A single-cell suspension was prepared by gently pipetting the cells up and down and incubating them for 30 minutes in the dark. The treated cells were analyzed by flow cytometry (BD LSRFortessa Cell Analyzer, BD Biosciences).
Reactive oxygen species generation was assessed using a ROS Detection Assay Kit following the manufacturer's instructions. HPDE6-C7 cells from different groups were collected, and the cell pellets were resuspended in culture medium containing 1 × ROS label. A single-cell suspension was prepared by gently pipetting the cells up and down and incubating them for 30 minutes in the dark. The treated cells were analyzed by flow cytometry (BD LSRFortessa Cell Analyzer, BD Biosciences).
6
Apoptosis and Cell Cycle Analysis by Flow Cytometry
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For apoptosis analyses, cells were seeded and infected with the retrovirus. After 96 h, trypsinized cells were collected and washed with cold PBS and resuspended in 1× Annexin V binding buffer (BD Biosciences) at a concentration of 1 × 106 cells/ml. After staining with propidium iodide and V450 annexin V (BD Biosciences), flow cytometric analyses typically using 10,000 cells were carried out using BD LSRFortessa cell analyzer. For cell cycle analyses, cells were seeded in 6-well plates and infected with the retrovirus. After 48 h, trypsinized cells were washed with cold PBS and fixed with 70% ethanol overnight. Subsequently, cells were stained with 50 µg/ml propidium iodide (PI; Sigma, USA) in PBS containing 10 μg/ml RNase A (Sigma) and 0.1% Triton X-100 at RT for 15 min. Flow cytometry for cell cycle analysis was carried out using BD LSRFortessa cell analyzer (BD Biosciences) and the distribution of a total of 10,000 nuclei was determined using BD FACSDiva software (BD Biosciences).
7
Cell Apoptosis and Cell Cycle Analysis
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The detection of cell apoptosis was performed utilizing a kit for apoptosis detection, specifically the Annexin V-FITC/PI obtained from Meilun in Dalian, China. Different culture media were added to 6-well plates to culture MDA-MB-231 cells for 48 h, which were initially treated with 0.25% trypsin and then rinsed twice with a PBS solution. Next, the gathered cells were treated with 5 µL of Annexin V-FITC and 5 µL of PI, which were then left to incubate at room temperature in the absence of light for a duration of 15 min. The stained cells were analyzed using flow cytometry (BD LSRFortessa™ Cell Analyzer; Franklin Lake, USA).
The measurement of the cell cycle was conducted by employing a cell cycle assay kit (Meilun; Dalian, China) in accordance with the guidelines provided by the manufacturer. MDA-MB-231 cells were collected, rinsed with PBS, and immobilized in ethanol for the duration of a whole night at 4 °C. Before analysis, the cells were rinsed once more with PBS, suspended and subjected to RNase A treatment with 10 µL for 30 min at 37 °C, and were subsequently incubated in the absence of light with 25 µL of PI for 30 min. Afterwards, the samples underwent an analysis using flow cytometry (BD LSRFortessa™ Cell Analyzer; Franklin Lake, USA).
The measurement of the cell cycle was conducted by employing a cell cycle assay kit (Meilun; Dalian, China) in accordance with the guidelines provided by the manufacturer. MDA-MB-231 cells were collected, rinsed with PBS, and immobilized in ethanol for the duration of a whole night at 4 °C. Before analysis, the cells were rinsed once more with PBS, suspended and subjected to RNase A treatment with 10 µL for 30 min at 37 °C, and were subsequently incubated in the absence of light with 25 µL of PI for 30 min. Afterwards, the samples underwent an analysis using flow cytometry (BD LSRFortessa™ Cell Analyzer; Franklin Lake, USA).
8
Mitochondrial Function, Glucose Uptake, and Calcium Dynamics
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Mitotracker assay—After treatments, 250,000 BT474 and BT474R cells were stained with 2 μM of JC-1 (MitoProbe JC-1 Assay kit for flow cytometry, Invitrogen) in media containing 0.1% FBS for 15 min at 37 °C. Cells were washed with cold PBS containing 1% FBS and analyzed on a Becton Dickinson (BD, Franklin Lakes, NJ, USA) LSRFortessa Cell Analyzer. Data were processed using BD FACSDiva software v 8.0.2.
Glucose uptake assay—After treatment, 300,000 BT474 and BT474R cells were incubated with 100 μM of 2-NBDG (Invitrogen) in DMEM (no glucose) containing 0.1% FBS for 30 min at 37 °C. Cells were washed with cold PBS containing 1% FBS and resuspended in wash buffer containing 0.3 μg/mL of DAPI (Invitrogen). Samples were analyzed on a BD LSRFortessa Cell Analyzer. Data were processed using BD FACSDiva software.
Intracellular calcium assay—After treatment, BT474 and BT474R cells were plated in a 96-well plate (20,000 cells/well) and loaded with Fluo-4 Direct from the Calcium Assay kit (Invitrogen) for 1 h at 37 °C. Fluorescence was measured at 494 nm using a Cytation 3 plate reader (Biotek, Winooski, VT, USA).
Glucose uptake assay—After treatment, 300,000 BT474 and BT474R cells were incubated with 100 μM of 2-NBDG (Invitrogen) in DMEM (no glucose) containing 0.1% FBS for 30 min at 37 °C. Cells were washed with cold PBS containing 1% FBS and resuspended in wash buffer containing 0.3 μg/mL of DAPI (Invitrogen). Samples were analyzed on a BD LSRFortessa Cell Analyzer. Data were processed using BD FACSDiva software.
Intracellular calcium assay—After treatment, BT474 and BT474R cells were plated in a 96-well plate (20,000 cells/well) and loaded with Fluo-4 Direct from the Calcium Assay kit (Invitrogen) for 1 h at 37 °C. Fluorescence was measured at 494 nm using a Cytation 3 plate reader (Biotek, Winooski, VT, USA).
9
Endogenous CD4 T cell responses and B cell costimulatory molecules in infected chimera
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Endogenous CD4 T cell responses in infected chimera were analyzed as described previously (Nothelfer et al., 2015) . Briefly, splenocytes were either restimulated with bone marrow-derived dendritic cells (BMDCs) pulsed with fixed parasites or phorbol 12-myristate 13-acetate (PMA)/ionomycin in the presence of Brefeldin A (BD Biosciences). Cells were then stained with anti-CD4-fluorescein isothiocyanate (FITC), anti-CD8 Pacific Blue, anti-IFN-g-allophycocyanin, and anti-IL-10-phycoerythrin (BD Biosciences). 350,000 cells were acquired on a BD LSRFortessa cell analyzer (Becton Dickinson), and analysis was performed using FlowJo software (Tree Star).
The expression of costimulatory molecules by B cells from infected chimeric mice was assessed using the following antibodies: FITC-conjugated anti-major histocompatibility complex class II (MHCII) (BD Biosciences), eFluor 450-conjugated anti-CD19 (eBioscience), PE-conjugated anti-CD40 (eBioscience), PE-conjugated anti-CD80 (eBioscience), and PE-conjugated anti-CD86 (eBioscience). Cells were acquired with a BD LSRFortessa cell analyzer (Becton Dickinson), and analysis was performed using FlowJo software (Tree Star).
The expression of costimulatory molecules by B cells from infected chimeric mice was assessed using the following antibodies: FITC-conjugated anti-major histocompatibility complex class II (MHCII) (BD Biosciences), eFluor 450-conjugated anti-CD19 (eBioscience), PE-conjugated anti-CD40 (eBioscience), PE-conjugated anti-CD80 (eBioscience), and PE-conjugated anti-CD86 (eBioscience). Cells were acquired with a BD LSRFortessa cell analyzer (Becton Dickinson), and analysis was performed using FlowJo software (Tree Star).
10
Optimizing Cell Line Pooling for Experimental Studies
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ZR-75-1, SKBR3, HCC-38, MDA-MB-231, and BT-20 cells were transduced with pHIV-Luc-ZsGreen (Addgene, Plasmid #39196) or pUltra-Chili-Luc (Addgene, Plasmid #48688) and sorted for GFP+ or RFP+ cells, as appropriate.
For preparing the five original pools, fluorescently labeled cell lines were counted, pooled, and frozen in liquid nitrogen. Pools were thawed on Day 0 and cultured normally. Six days and 13 days after thawing, the percentages of GFP+ and RFP+ cells were quantified by flow cytometry analysis using a LSRFortessa Cell Analyzer (BD Biosciences).
For estimating the growth rate (r) of each of the five cell lines, the equation for exponential cell growth was used: where xt = percentage at day t, x0 = percentage at day 0, r = growth rate, and t = time [days]. Growth rates (r) were then used to calculate the optimal starting percentage (x0) of each cell line to achieve 20% representation at t = 7 days.
For preparing the five optimized pools, fluorescently labeled cell lines were counted, pooled according to our model, and frozen in liquid nitrogen. Pools were thawed on Day 0 and cultured normally. Seven days after thawing, the percentages of GFP+ and RFP+ cells were quantified by flow cytometry analysis using a LSRFortessa Cell Analyzer (BD Biosciences). Heat maps were generated using Cluster 3.0 and Java TreeView 1.1.6r4.
For preparing the five original pools, fluorescently labeled cell lines were counted, pooled, and frozen in liquid nitrogen. Pools were thawed on Day 0 and cultured normally. Six days and 13 days after thawing, the percentages of GFP+ and RFP+ cells were quantified by flow cytometry analysis using a LSRFortessa Cell Analyzer (BD Biosciences).
For estimating the growth rate (r) of each of the five cell lines, the equation for exponential cell growth was used: where xt = percentage at day t, x0 = percentage at day 0, r = growth rate, and t = time [days]. Growth rates (r) were then used to calculate the optimal starting percentage (x0) of each cell line to achieve 20% representation at t = 7 days.
For preparing the five optimized pools, fluorescently labeled cell lines were counted, pooled according to our model, and frozen in liquid nitrogen. Pools were thawed on Day 0 and cultured normally. Seven days after thawing, the percentages of GFP+ and RFP+ cells were quantified by flow cytometry analysis using a LSRFortessa Cell Analyzer (BD Biosciences). Heat maps were generated using Cluster 3.0 and Java TreeView 1.1.6r4.
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