The C57BL/6 splenic cells were made into single cell suspensions. And the cells were stained with anti-CD4 FITC antibodies for 30 minutes. Then the cells were sorted by the S3TM Cell Sorter (Bio-Rad Laboratories, Inc.). The T cells and pretreated DCs or unpretreated DCs were seeded together in 96-well plates. After coincubation for 72 h, cell proliferation was measured with the CFDA SE Cell Proliferation Assay and Tracking Kit (Beyotime, #C0051, China) according to the manufacturer's protocol.
S3tm cell sorter
Manufactured by Bio-Rad
Sourced in United States
The S3TM Cell Sorter is a flow cytometry-based instrument designed for the separation and isolation of specific cell populations. It employs hydrodynamic focusing to align and analyze individual cells passing through a laser beam, allowing for the detection and sorting of target cells based on their distinct physical and fluorescent properties.
Automatically generated - may contain errors
Lab products found in correlation
Cell dissociation buffer, by Thermo Fisher Scientific (2 mentions)
Cfda se cell proliferation assay and tracking kit, by Beyotime (1 mentions)
Anti cd28 37.51, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
M1 70, by Thermo Fisher Scientific (1 mentions)
Rbc lysing buffer, by Merck Group (1 mentions)
Gentlemacs tissue dissociator, by Miltenyi Biotec (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Cd86 pe, by BioLegend (1 mentions)
Cd206 fitc, by Miltenyi Biotec (1 mentions)
Fvb n mice, by Jackson ImmunoResearch (1 mentions)
Propidium iodide solution, by Merck Group (1 mentions)
Rnase a, by Qiagen (1 mentions)
10 protocols using s3tm cell sorter
1
T Cell Proliferation Assay with DCs
2
Phenotyping Polarized Immune Cells
3
Analyzing CD8+ T cell proliferation
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Mouse CD3+ T cells were isolated from spleens using a S3TM Cell Sorter (Bio-Rad Laboratories, Hercules, CA). Sorted CD3+ T cells with the high purity (>95%) using an antibody (145-2C11, eBioscience) were labeled with carboxyfluorescein succinimidyl ester (CFSE) (2.5 μM; Invitrogen) according to the manufacturer’s guideline and plated at 1 × 105/well in 96-well plates containing 2 μg/ml anti-CD3 (145-2C11, eBioscience) and 2 μg/ml anti-CD28 (37.51, eBioscience) mAbs. After 3 days, the cells were stained with CD8 antibody (53-6.7, eBioscience), and then the CD8+ T cell proliferation was determined by flow cytometry at the PEC/T cell ratio of 1:2.
4
Isolation of GFP-labeled Hemocytes from Drosophila
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GFP-labeled hemocytes were isolated from HmlΔ-Gal4 UAS-eGFP male flies using fluorescence-activated cell sorting (FACS). Approximately 200 flies were anaesthetized with CO2, washed in PBS and homogenized in 600 μL of PBS using a pestle. Homogenate was sieved through a nylon cell strainer (⌀ 40 μm). This strainer was then additionally washed with 200 µL of PBS, which was added to the homogenate subsequently. Samples were centrifuged (3 min, 6°C, 3500 RPM) and the supernatant was washed in ice cold PBS after each centrifugation (3x). Prior to sorting, samples were transferred to polystyrene FACS tubes using a disposable bacterial filter (⌀ 50 µm, Sysmex) and sorted into 100 µL of TRIzol Reagent (Invitrogen) using a S3TM Cell Sorter (BioRad). Sorted cells were verified by fluorescent microscopy and by differential interference contrast (DIC).
5
Isolating GFP-expressing Macrophages from Flies
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As
a reference experiment,
the GFP-expressing macrophages were isolated from CrqGal4 > GFP
male
flies using fluorescence-activated cell sorting (FACS). Three hundred
flies were anesthetized with CO2, washed in PBS, and homogenized
in 600 mL of PBS using a pestle. The homogenate was sieved through
a nylon cell strainer (40 μm). This strainer was then additionally
washed with 200 μL of PBS, which was added to the homogenate
subsequently. The samples were centrifuged (3 min, 4 °C, 800g), and the supernatant was washed with ice-cold PBS after
each centrifugation (3 times). Prior to sorting, samples were transferred
to FACS polystyrene tubes using a disposable bacterial filter (50
μm, Sysmex), and macrophages were sorted into 100 μL of
PBS using a S3TM Cell Sorter (BioRad). Isolated cells were verified
by fluorescence microscopy and differential interference contrast.
a reference experiment,
the GFP-expressing macrophages were isolated from CrqGal4 > GFP
male
flies using fluorescence-activated cell sorting (FACS). Three hundred
flies were anesthetized with CO2, washed in PBS, and homogenized
in 600 mL of PBS using a pestle. The homogenate was sieved through
a nylon cell strainer (40 μm). This strainer was then additionally
washed with 200 μL of PBS, which was added to the homogenate
subsequently. The samples were centrifuged (3 min, 4 °C, 800g), and the supernatant was washed with ice-cold PBS after
each centrifugation (3 times). Prior to sorting, samples were transferred
to FACS polystyrene tubes using a disposable bacterial filter (50
μm, Sysmex), and macrophages were sorted into 100 μL of
PBS using a S3TM Cell Sorter (BioRad). Isolated cells were verified
by fluorescence microscopy and differential interference contrast.
6
Differentiation of Murine CD11b+ Cells
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Bone marrow cells were obtained from the femurs of Balb/c mice. BM cells were treated with RBC lysing buffer (Sigma-Aldrich, St. Louis, MO, USA) for depletion of red blood cells. Next, the remaining cells were stained with an antibody against CD11b (M1/70, eBioscience, San Diego, CA, USA) for CD11b+ cell sorting using a S3TM Cell Sorter (Bio-Rad Laboratories, Hercules, CA, USA). The sorted CD11b+ cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS in the presence of 10 ng/ml GM-CSF, in the absence or presence of 50 ng/ml LPS and TCCM in an atmosphere of 5% CO2 in a 37°C humidified incubator. The cells were collected on day 4 for cell analysis.
7
Rapid Autopsy Tissue Analysis
8
Macrophage Immunophenotyping by Flow Cytometry
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Cells were dissociated using enzyme-free Cell Dissociation Buffer (13151014, ThermoFisher) with scraping. In vitro human macrophages were stained with CD206-FITC (130-100-085, Miltenyi Biotec), CD163-PE-Cy7 (clone GHI/61, 333614, BioLegend), CD86-PE (305406, BioLegend), and propidium iodide (PI, 00-6990-50, eBioscience). Data was collected using a BioRad S3TM Cell Sorter and analysis was performed using FlowJoⓇ.
9
Genotyping and Validating IL-4Ra Knockout Mice
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The Johns Hopkins Institutional Animal Care and Use Committee approved all experiments involving mice (protocol # MO19M41). FVB/N mice were purchased from The Jackson Laboratory (Bar Harbor, ME). FVB/N Il4raem1/em1 (Il4ra KO) mice were created by The Jackson Laboratory (Stock No. 037518) by whole animal gene knockout using CRISPR/Cas9 to remove exon 4 of Il4ra. Genotyping was performed by tail snip DNA extraction and PCR using forward primer 5’- AGCCTGAGCCGTACAGATTG-3’ (common) and reverse primers 5’-ACAGAACGGCCAGATCAGTG-3’ (WT) and 5’- TAACAGAACGCAGGGTCATC-3’ (Mutant). To confirm IL-4R alpha protein knockout, mouse spleen cells were stained with anti-mouse CD124-PE (clone mIL4R-M1, 561695, BD Biosciences) or isotype rat IgG2a,κ-PE (553930, BD Biosciences) and PI. Data was collected using a BioRad S3TM Cell Sorter and analysis was performed using FlowJoⓇ.
10
Cell Cycle Analysis of hESCs
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For cell cycle analysis, 35 000 cells were seeded per well in duplicates in complete naive media either supplemented with 150µM HU or equal amounts of PBS. Cells were trypsinized and MEFs were depleted as described above. Loosened hESC colonies and remaining MEF were resuspended in complete hESC media and centrifuged at 39g. Next pellets were washed with PBS followed by a hESC specific staining by adding 0.5µl of the TRA-1-60-FITC antibody (560173, BD Pharmingen) per 100 000 cells and incubated for 30min at 4°C in the dark. To avoid overstaining, cells were washed in PBS and were subsequently resuspended in 300µl cold PBS and, while vortexing, 700µl ice-cold ethanol was added dropwise to fix the cells. Following an incubation of the sample for minimum 1h at -20°C, cells were washed in PBS and resuspended in 100µl PBS with RNase A (19101, Qiagen) to a final concentration of 0.25mg/ml. Upon 1h incubation at 37°C, 4µl propidium iodide solution (P4170-25mg, Sigma)
were added to a final concentration of 40µg/ml. Samples were loaded on a BioRad S3TM cell sorter and analysed with the Dean-Jett-Fox algorithm for cell-cycle analysis using the ModFit LTTM software package. Error bars in figures represent SD after scaling to control and error propagation. For statistical testing, a Wilcoxon rank sum test was performed at the 5% significance level.
were added to a final concentration of 40µg/ml. Samples were loaded on a BioRad S3TM cell sorter and analysed with the Dean-Jett-Fox algorithm for cell-cycle analysis using the ModFit LTTM software package. Error bars in figures represent SD after scaling to control and error propagation. For statistical testing, a Wilcoxon rank sum test was performed at the 5% significance level.
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