PBMCs isolated from healthy donors were plated at 1×106 cells/mL in RPMI1640 plus 10% fetal calf serum, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine, 10 ng/mL GM-CSF and IL-6 at 37°C at 10% CO2 for 7 days with half medium refresh every other day. CD33+MDSCs were then enriched by anti-CD33 mcirobeads (Miltenyi Biotec). 100 nM siRNAs against CCRK (siCCRK: 5'-GGCGGUUGGAGGACGGCUU-3'), and a control sequence (siCtrl: 5'-UUCUCCGAACGUGUCACGU-3')4 (link) were transfected into the purified CD33+MDSCs by Neon™ Transfection System (ThermoFisher) according to the manufacturer’s protocols. Cells were collected 24-hour post-transfection for RNA extraction and 48 h post-transfection for protein isolation. For MDSC proliferation assay, carboxyfluorescein succinimidyl ester (CFSE; 5 μmol/L; Invitrogen) was used to label purified CD33+MDSCs before siRNA treatment. Cells were collected at 72-hour post-transfection and CFSE signals were acquired by flow cytometry using FACSAria Fusion (BD Biosciences). For phenotype analysis, CD33+MDSCs treated with siCtrl or siCCRK were collected at 48-hour post-transfection, and analysed by flow cytometry using FACSAria Fusion (BD Biosciences). The antibodies used are listed in the key resources table .
Facsaria fusion
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The FACSAria Fusion is a high-performance cell sorter designed for advanced flow cytometry applications. It features a compact, modular design and offers a range of configuration options to meet the specific needs of researchers.
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Lab products found in correlation
Facsaria 2, by BD (48 mentions)
Lsrfortessa, by BD (39 mentions)
Facsaria 3, by BD (32 mentions)
Facscanto 2, by BD (26 mentions)
Facsdiva software, by BD (22 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
Facsdiva, by BD (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Lsrfortessa x 20, by BD (12 mentions)
Novaseq 6000, by Illumina (11 mentions)
Facsaria, by BD (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (10 mentions)
Fortessa, by BD (10 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Facsmelody, by BD (8 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (7 mentions)
Facscalibur, by BD (7 mentions)
Dnase 1, by Merck Group (7 mentions)
Ionomycin, by Merck Group (7 mentions)
Attune nxt, by Thermo Fisher Scientific (7 mentions)
833 protocols using facsaria fusion
1
Isolation and Silencing of Human MDSCs
2
CRISPR-Mediated Genetic Modifications in HeLa Cells
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The sequences of gRNAs are listed in Appendix Table S2. For single knockout clones, annealed gRNAs of target genes were ligated into the pX458 vector. The plasmids were transiently transfected into HeLa cells using PEI as the transfection reagent. Two days later, GFP-positive cells were sorted into 96-well plate (one cell per well) by flow cytometry with the BD FACSAria Fusion. Single clones were grown for 2-3 weeks, and the resultant colonies were expanded and examined by immunoblotting and sequencing. For pool knockout, annealed gRNAs were ligated into the lentiCRISPR v2 vector. The plasmids were used to package lentivirus to infect the targeted cells as described previously. Infected cells were selected by 2 μg/ml puromycin for 48 h, and the knockout efficiency was determined by immunoblotting. For FLAG knockin cells, annealed gRNAs of target genes were also cloned into the pX458 vector. The HMEJ donor containing the FLAG sequence flanked by 800-bp homology arms complementary to the knockin site with PAM mutation was inserted into pBM16A T-vector (Yao et al, 2017) . For each well of a six-well plate, a total of 2 μg plasmids (pX458:donor = 1:1) was used. After 48 h, GFP-positive HeLa cells were sorted into 96-well plates (one cell per well) by flow cytometry with the BD FACSAria Fusion. FLAG knockin clones were examined by immunoblotting and confirmed by DNA sequencing.
3
Single-cell sorting for epigenetic analysis
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TSC WT cells (TSC1 and TSC2) were sorted as single cells onto MEF-coated 96-well plates containing TSC medium +FGF4 (37.5 ng ml−1), Heparin (1.5 µg ml−1) and 1× ROCKi using the BD FACSAria Fusion instrument.
ESC WT cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium (Knockout DMEM (Thermo Fisher Scientific, #10829018, 15% foetal bovine serum (PAN, #P30-2602), 1× GlutaMAX supplement (Thermo Fisher Scientific, #35050-038), 1× non-essential amino acids (Thermo Fisher Scientific, #11140-035), 100 µM 2-mercaptoethanol (Thermo Fisher Scientific, #21985023), 1× penicillin–streptomycin (Thermo Fisher Scientific, #15140122) and lab-purified recombinat leukaemia inhibitory factor (LIF) using the BD FACSAria Fusion instrument. Cells were then expanded and MEF-depleted for methylation analysis by RRBS.
ESC WT cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium (Knockout DMEM (Thermo Fisher Scientific, #10829018, 15% foetal bovine serum (PAN, #P30-2602), 1× GlutaMAX supplement (Thermo Fisher Scientific, #35050-038), 1× non-essential amino acids (Thermo Fisher Scientific, #11140-035), 100 µM 2-mercaptoethanol (Thermo Fisher Scientific, #21985023), 1× penicillin–streptomycin (Thermo Fisher Scientific, #15140122) and lab-purified recombinat leukaemia inhibitory factor (LIF) using the BD FACSAria Fusion instrument. Cells were then expanded and MEF-depleted for methylation analysis by RRBS.
4
BrdU and pS10-H3 Fluorescence-Activated Cell Sorting Protocol
5
Isolating ILC1s and NK Cells from Mouse Liver and Human Blood
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To isolate ILC1s or NK cells from mouse liver, we washed harvested liver and pressed it through a 100 μm mesh to make single cells, which were washed once with PBS. The cells were re-suspended in 40% Percoll (Sigma-Aldrich) and then gently overlaid on 70% Percoll, followed by centrifugation according to the manufacturer’s instructions. Mononuclear cells (MNCs) were collected from the interphase and washed twice with PBS. The washed MNCs were stained with anti-CD3, anti-CD19, anti-NK1.1, anti-NKp46, anti-CD49b, and anti-CD49a antibodies. Thirty minutes later, the cells were washed 3 times and then sorted using BD FACSAria™ Fusion.
To isolate ILC1s from human peripheral blood, we diluted blood cone samples 1:1 with phosphate-buffered saline (PBS). We layered the blood on the top of Ficoll-Paque (GE Healthcare), and centrifuged it according to the manufacturer’s instructions. The mononuclear cell fraction was aspirated and washed with PBS, and then the red blood cells were lysed. The mononuclear cells were stained with lineage (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD20, anti-CD33, anti-CD34, anti-CD203c, anti-FceRI, and anti-CD56), anti-CD127, anti-CRTH2, and anti-c-Kit antibodies. Thirty minutes later, the cells were washed 3 times and then sorted using BD FACSAria™ Fusion.
To isolate ILC1s from human peripheral blood, we diluted blood cone samples 1:1 with phosphate-buffered saline (PBS). We layered the blood on the top of Ficoll-Paque (GE Healthcare), and centrifuged it according to the manufacturer’s instructions. The mononuclear cell fraction was aspirated and washed with PBS, and then the red blood cells were lysed. The mononuclear cells were stained with lineage (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD15, anti-CD16, anti-CD19, anti-CD20, anti-CD33, anti-CD34, anti-CD203c, anti-FceRI, and anti-CD56), anti-CD127, anti-CRTH2, and anti-c-Kit antibodies. Thirty minutes later, the cells were washed 3 times and then sorted using BD FACSAria™ Fusion.
6
Isolating ILC1s and NK Cells from Mouse Liver and Human Blood
7
Identification of HIV-gp140 Memory B Cells
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The HIV-specific memory B cells were identified using the HIV-gp140 protein probe as per the previously reported protocol (25 (link), 26 (link)) with some modifications. In brief, after revival and resting, the PBMCs from the study participants were stained with CD3 BV786, CD19 FITC, and CD27 PE (all from BD Biosciences) and with biotinylated gp140 (5 µg/ml) for 30 min at room temperature and were then stained with streptavidin PE-Cy7 (BD Biosciences) for 30 min at room temperature and acquired on BD FACSAria™ Fusion (BD Biosciences). The total memory B cells were gated as CD3−CD19+CD27+ cells from lymphocytes (Figure 2A Figure 2C
8
Paclitaxel-Induced Apoptosis in SKOV3-TR30 Cells
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SKOV3-TR30 cells were plated in 6-well plates (2 × 105 cells/2 mL medium). 48 h after transfection, the cells were treated with 200 nmol/L paclitaxel. After an additional 24 h, the cells were harvested and washed twice in PBS for the detection of cell apoptosis using an Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, Mountain View, CA, USA) according to the manufacturer's instructions. The cells were stained with Annexin V and PI in the binding buffer for 30 min in the dark and analyzed by flow cytometry (BD FACSAria TM Fusion). Approximately 104 cells were estimated per sample, and three independent experiments were performed.
9
Isolation and Characterization of Cardiac Immune Cells
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To study immune cell infiltrates in the postnatal heart, heart tissues were minced into small fragments and dissociated with 1:1 type II collagenase (1000 U/ml in PBS, Worthington) and dispase (11 U/ml in PBS, Gibco) at 37°C for 30 min. Enzymatic action was stopped by adding 10% FBS and the dissociated cells were washed twice with PBS. The dissociated single splenocytes or neonatal heart cells were removed from the contaminated erythrocytes by incubating with the red blood cell lysis buffer (eBiosciences) for 5 min; and were then blocked with 2% normal rabbit serum. Cells were subsequently stained with fluorochrome-conjugated antibodies against the following antigens: CD3, CD4, CD8, CD45, CD206, F4/80 or Ly6C (Biolegend or eBiosciences) at a dilution of 1:100, unless specified by the manufacturer, at 4°C for 30 min. Murine Treg of ICR mice were detected with the Treg staining kit according to manusfacturer's instructions (eBioscience). Cells were then washed three times with 2% FBS-containing PBS and analyzed on flow cytometer (BD FACSAriaTM Fusion). Propidium iodide (PI, BD) positive dead cells were excluded for live cell analysis/sorting; and FACS data were then analyzed with the FlowJo software (Tree star).
10
Multiparameter Flow Cytometry Analysis
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Cells were detached from the culture plates by treatment with 0.05% trypsin-1% EDTA (Gibco) and collected by a cell scraper (Iwaki), centrifuged, resuspended in wash buffer (2%FBS in PBS), filtered using 100-μm mesh (Falcon), and counted by a Countess cell counter (ThermoFisher). Cells (0.1–1.0 × 107) were incubated with antibodies (1:200 dilutions) in 700 μl PBS containing 2% FBS (wash buffer) for 30 min at 4 °C, washed in wash buffer, incubated with propidium iodide briefly to stain dead cells, and analyzed and sorted using a BD FACSAriaTM Fusion equipped with three lasers (405, 561, 640 nm) using FACS Diva (BD Bioscience, v 8.0) and FlowJo (BD Biosciences). The following antibodies were used in this study: CD57(HNK-1)-PE (clone TB03, Miltenyi Biotec), ERBB3-APC (clone REA508, Miltenyi Biotec), CD271-BB515 (clone C40-1457, BD Pharmingen), and human NOTCH3-PE (clone: MHN3-21, BioLegend).
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