Primary B cells isolated from the spleen/LN (sorted for CD19+ or B220+) were stimulated with LPS (1 µg/ml). CD4+ T cells (>98%) from the spleen and/or LN were activated in plate-bound anti-CD3 Abs (10 µg/ml) and soluble anti-CD28 Abs (1 µg/ml) as recommended by the manufacturer (BD Pharmingen, San Diego, CA, USA) and as previously described25 (link). For intracellular cytokine detection, cells were re-stimulated for 5 h with PMA (Sigma, P8139) (20 ng/ml) and ionomycin (Sigma I0634) (1 µM). Golgi-stop was added in the last hour and intracellular cytokine staining was performed using BD Biosciences Cytofix/Cytoperm kit as recommended (BD Pharmingen, San Diego, CA, USA). FACS analysis was performed on a Becton-Dickinson FACSCalibur (BD Biosciences) using protein-specific monoclonal antibodies and corresponding isotype control Abs (PharMingen, San Diego, CA, USA) as previously described13 (link), 25 (link). FACS analysis was performed on samples stained with mAbs conjugated with fluorescent dyes and each experiment was color-compensated. Dead cells were stained with dead cell exclusion dye (Fixable Viability Dye eFluor 450; eBioscience) and live cells were subjected to side-scatter (SSC) and forward scatter (FSC) analysis. Quadrant gates were set using isotype controls with less than 0.2% background.
Becton dickinson facscalibur
Manufactured by BD
Sourced in United States
The Becton Dickinson FACSCalibur is a flow cytometry instrument. It is designed to analyze and sort cells based on their physical and fluorescent characteristics. The FACSCalibur can measure multiple parameters simultaneously on individual cells within a sample.
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Lab products found in correlation
Ionomycin, by Merck Group (2 mentions)
Hepes, by MP Biomedicals (2 mentions)
70 μm strainer, by BD (2 mentions)
Fcs express 4, by De Novo Software (2 mentions)
Anti cd3 ab, by BD (1 mentions)
Fixable viability dye efluor 450, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Modfitlt version 3, by Verity Software House (1 mentions)
Polybrene, by Merck Group (1 mentions)
Apc cyanine7 anti mouse cd86, by BioLegend (1 mentions)
Cd11b bv421, by BioLegend (1 mentions)
Cd86 pe, by BD (1 mentions)
Cd11c percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd206 fitc, by BioLegend (1 mentions)
Cd45 apc, by BD (1 mentions)
Pe conjugated anti gr 1, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti cd71, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti sca 1, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
15 protocols using becton dickinson facscalibur
1
Isolation and Stimulation of Primary B and T Cells
2
Cell Cycle Analysis of Sterol Starved Cells
3
Lentiviral Transduction of Cardiac Progenitor Cells
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The bicistronic LV-HCN4 expression vector has previously been described [32 (link)]. Briefly, HCN4 expression is controlled by a cytomegalovirus (CMV) promoter and linked to green fluorescent protein (GFP) expression by an internal ribosome entry site from encephalomyocarditis virus. LV vectors with CMV-driven GFP and DsRed genes served as control vectors. The vectors are designated LV-HCN4-GFP, LV-GFP, and LV-DsRed, respectively. LV particles were generated by co-transfection of HEK293T cells, and concentrated and titrated as previously described [50 (link)].
CMPCs were transduced in the presence of 8 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA, cat.no. TR-1003), with LV-HCN4-GFP or LV-GFP at an MOI of 2. Transduced cells were used after 4 days for co-culture and organ explant experiments, or within 7–14 days for patch-clamp analysis. To assess the LV transduction efficiency, 5 × 104 CMPCs were transduced as above with LV-DsRed at MOIs of 1, 2, and 10. The cells were washed twice in phosphate-buffered saline (PBS), trypsinized, resuspended in SP++ medium, and analyzed using a Becton Dickinson FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA).
CMPCs were transduced in the presence of 8 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA, cat.no. TR-1003), with LV-HCN4-GFP or LV-GFP at an MOI of 2. Transduced cells were used after 4 days for co-culture and organ explant experiments, or within 7–14 days for patch-clamp analysis. To assess the LV transduction efficiency, 5 × 104 CMPCs were transduced as above with LV-DsRed at MOIs of 1, 2, and 10. The cells were washed twice in phosphate-buffered saline (PBS), trypsinized, resuspended in SP++ medium, and analyzed using a Becton Dickinson FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA).
4
Flow Cytometry of Microglial Antigens
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Cells were assayed for surface antigens by flow cytometry as previously described (43 (link)). These microglia were labeled with fluorescein isothiocyanate anti-mouse CD45 (2 μg/ml; BioLegend, California, USA), APC (Allophycocyanin) anti-mouse CD11b (5 μg/ml; BioLegend, California, USA), APC/Cyanine7 anti-mouse CD86 (1 μg/ml; BioLegend, California, USA), and PerCP/Cyanine 5.5 CD206 (5 μg/ml; BioLegend, California, USA) antibodies for 30 min at room temperature. A four-laser Becton-Dickinson FACS Calibur (BD Biosciences, New Jersey, USA) was used to collect the data, and FlowJo software was used for analysis.
5
Corneal Cell Isolation and Characterization
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The corneas were dissected out and placed in 1 ml of RPMI with deoxyribonuclease І (0.002 g) and collagenase D (0.004 g) in a 37°C bath for 60 min, with inverting every 10 min while pipetting up and down. The digestion was stopped by 200 μl of FBS, and cell suspension was filtered through a 70-μm strainer (BD Biosciences, San Diego, CA, USA). The cells were washed with PBS containing 1% FBS and 1% Hepes (MP Biomedicals) and stained with CD45-APC (559864, BD Pharmingen), CD11b-BV421 (101236, BioLegend), CD11c-PerCP-Cy5.5 (45-0114-83, eBioscience), CD86-PE (553629, BD Pharmingen), and CD206-FITC (141704, BioLegend) antibodies for 30 min at room temperature. A multilaser Becton-Dickinson FACSCalibur (BD Biosciences) was used to collect the data, and FlowJo software was used for analysis.
6
Monoclonal Phage-scFv Binding Assay
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Binding reactivity of the monoclonal phage-scFvs was further verified by cell-based ELISA and flow cytometry using AGS, MKN-45, and NIH-3T3 cells. To this end, phage-scFvs of 4 individual clones were produced in large culture volumes of 2xTY-Amp-Glc medium and purified by PEG (polyethylene glycol) precipitation. For flow cytometry analysis, the cells and the phage-scFvs were blocked in FACS buffer (PBS solution containing 3% BSA and 0.03% NaN3) and aliquots containing 5x105 (link) cells were incubated with 100 µL of phage particles (5×1011 CFU) for 1 h on ice. The membrane-bound phage particles were subsequently detected by incubation with anti-M13 monoclonal antibody and FITC-conjugated rat anti-mouse IgG (BioLegend, San Diego, California, USA). All incubations were performed at 4°C followed by two washes with PBS containing 1% BSA and 0.03% NaN3. After the final wash and resuspension in 500 µL PBS, the samples were investigated through Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) while dead cells were excluded by adding 5 µL of propidium iodide.
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Binding reactivity of the monoclonal phage-scFvs was further verified by cell-based ELISA and flow cytometry using AGS, MKN-45, and NIH-3T3 cells. To this end, phage-scFvs of 4 individual clones were produced in large culture volumes of 2xTY-Amp-Glc medium and purified by PEG (polyethylene glycol) precipitation. For flow cytometry analysis, the cells and the phage-scFvs were blocked in FACS buffer (PBS solution containing 3% BSA and 0.03% NaN3) and aliquots containing 5x105 (link) cells were incubated with 100 µL of phage particles (5×1011 CFU) for 1 h on ice. The membrane-bound phage particles were subsequently detected by incubation with anti-M13 monoclonal antibody and FITC-conjugated rat anti-mouse IgG (BioLegend, San Diego, California, USA). All incubations were performed at 4°C followed by two washes with PBS containing 1% BSA and 0.03% NaN3. After the final wash and resuspension in 500 µL PBS, the samples were investigated through Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) while dead cells were excluded by adding 5 µL of propidium iodide.
7
Immunophenotyping of Hematopoietic Cell Populations
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Freshly isolated cells were washed twice in PBS (Gibco) and immunostained for 15 min with the appropriate antibody; phycoerythrin (PE)-conjugated anti-TER119 (erythroid marker), PE-conjugated Anti-cKIT (SCF receptor), PE-conjugated anti-CD41 (glycoprotein IIb), PE-conjugated anti-CD61 (glycoprotein IIIa), PE-conjugated anti-Gr-1 (granulocytic marker), PE-conjugated anti-MAC-1 (monocytic marker), APC-conjugated anti-CD71 (transferrin receptor), APC-conjugated anti-SCA-1 (primitive hematopoietic cell marker) (1:200) (eBioscience, San Diego, CA, USA). Following antibody incubation, cells were washed once in PBS and resuspended in 500 μl PBS. Cell sorting and analysis of stained cells were performed using the Becton-Dickinson FACSCalibur (BD Biosciences, San Jose, CA, USA), and the FlowJo flow cytometry analysis software (FlowJo TreeStar Inc., Ashland, OR, USA). Relative mean fluorescence intensity (MFI) values were based on the unstained population controls and calculated using the Geometric Mean statistic (average of log fluorescence). Statistical analyses were performed using the two-tailed Student's t-test, where P<0.05 was considered to indicate a statistically significant difference.
8
Retinal Immune Cell Profiling in Mice
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The eyeballs were enucleated from C57 and rd1 mice at P14, P21, P28, and P180. Retinae were dissected out and placed in 1 ml RPMI with a pH of 7.4 regulated by Hepes (1:100, MP BIOMEDICALS), and disaggregated by gently pipetting up and down through a wide bore pipette tip as previously described (Noailles et al., 2016 (link)). This cell suspension was filtered through a 70-μm strainer (BD Biosciences, San Diego, CA, United States) to prevent cell clumps. The cells were washed with phosphate-buffered saline (PBS) containing 1% FBS (Life Technologies, Grand Island, NY, United States) and 1% Hepes (MP BIOMEDICALS), and were stained by CD45 (2 ug/ml, BD pharmingenTM), CD11b (5 ug/ml, eBioscience), CD86 (1 ug/ml, BD pharmingenTM), and CD206 (5 ug/ml, Biolegend) antibodies for 30 min at room temperature. A four-laser Becton-Dickinson FACS Calibur (BD Biosciences) was used to collect the data, and FlowJo software was used for analysis.
9
Cellular Oxidative Stress Measurement
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For eight weeks, cells were grown under NG or HG conditions and incubated for 48 h with Exe-4 (10 nM) and/or DDP (25 µM). After washing with PBS, the cells were collected and resuspended at 1×106/ml, incubated with DCFH-DA (KeyGEN BioTECH; 10 μM) for 20 min at 37°C. Then, the cells were washed with a serum-free cell culture medium three times. Cellular ROS levels were detected by flow cytometry on a Becton-Dickinson FACS Calibur (BD Biosciences, Franklin Lake, NJ, USA).
10
Macrophage Phenotypic Maturation Assay
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For a 24 hour period prior to collection, 1 x 106 macrophage/mL were incubated with PBS, 50 μg/mL of PLGA NPs, and 50 μg/mL PLGA/OVA/MPLA NP in individual standard wells of a 12-well plastic culture plate. Three wells were assigned to each of the aforementioned study groups. The macrophages treated with PBS were utilized as a control. The cells were collected using a sterile cell scraper, and gently washed using PBS at 37°C. For phenotypic maturation studies, the macrophages of each group were separately incubated with 15 μg/mL of COLIS69A (mAb isotype control with specificity for E.coli J5), MHC-I (DH-H58A) and MHC-II (TH14B), followed by incubation with a fluorescein-conjugated IgG/IgM antibody. All mAbs were obtained from the Washington State University Monoclonal Antibody Center, Pullman, WA. Samples were acquired on a Becton Dickinson FACSCalibur (BD Biosciences, Franklin Lakes, NJ) by gating on the live cell populations. All non-CD14+ populations were excluded. The data was further evaluated using FCS Express 4 (DeNovo Software) and presented as dot plots.
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