Adipose tissue depots were pooled to maximize collection of APCs. SVF was extracted and stained with CD45, CD11c, CD64, and CD3 flow antibodies (listed above). A FACS sort was performed to isolate ATMs, ATDCs, CD11c- CD64- CD45+, and CD45- cell populations using a Sony MA900 sorter. CD3+ cells were excluded. Each of the 4 cell populations was plated overnight, before being pulsed with 10ug/ml of OVA or BSA for 6 hours. CD3+ cells were isolated from the lymph nodes and spleen of an OTII mouse. These cells were then stained with CFSE and co-cultured with APCs at a 1:10 ratio. After 4 days cells were taken and stained for flow cytometry analysis. Gating strategy is as follows: Singlets (FSC-W x FSC-H) → Scatter → Live cells (Live/Dead Fixable Violet Dead Cell Stain Kit, Invitrogen) → CD4+ → FoxP3− → CD25+ CFSE−
Ma900 sorter
Manufactured by Sony
Sourced in United States, Japan
The MA900 sorter is a high-performance laboratory equipment designed for efficient sorting and separation of various materials. It features advanced sorting mechanisms and precise control systems to ensure accurate and reliable results. The core function of the MA900 sorter is to separate and categorize different types of samples or materials based on specific physical or chemical properties.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll, by BD (2 mentions)
Pe anti human cd45, by BioLegend (2 mentions)
Apc anti human cd117, by BioLegend (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
C tube, by Miltenyi Biotec (2 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
C9263, by Merck Group (1 mentions)
Anti cd45 af700, by BD (1 mentions)
Collagenase type 5, by Merck Group (1 mentions)
100 μm mesh, by BD (1 mentions)
Soy trypsin inhibitor, by Thermo Fisher Scientific (1 mentions)
Octo dissociator, by Miltenyi Biotec (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Fc block, by BD (1 mentions)
Sh800 sorter, by Sony (1 mentions)
Gateway recombination, by Thermo Fisher Scientific (1 mentions)
Trustain fcx, by BioLegend (1 mentions)
21 protocols using ma900 sorter
1
Isolation and Functional Analysis of Adipose Tissue Immune Cells
2
Isolation of Live Tumor Cells
3
Tumor Cell Isolation and Flow Cytometry
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Tumor cell suspensions were generated by initial mechanical disaggregation/mincing. Tumor fragments were then transferred to a solution of type V collagenase (Sigma C9263, 1 mg ml−1 in 1× HBSS) supplemented with soy trypsin inhibitor (Gibco, 0.1 mg ml−1) and DNase I (Sigma, 0.1 mg ml−1). Tumor pieces in this disaggregation buffer were transferred to a GentleMACS tube and loaded into the OctoDissociator (Miltenyi). Samples were treated with the mTDK1 program, after which 5 ml of FACS buffer (PBS 1×, 2% FBS) was added to the sample and the mix was filtered through a 100-μm mesh (BD). The resulting cell suspension was centrifuged and resuspended in FACS buffer. Cells were then treated with Fc block (BD, 1:200 dilution) incubated at 4 °C for 15 min and stained with anti-CD45 AF700 (BD, 1:400 dilution) for 30 min at 4 °C. Cells were washed and resuspended in FACS buffer supplemented with DAPI (Sigma, 1 μg ml−1 final). Stained cell suspensions were then analyzed in an MA900 sorter (Sony). EGFP+ cells were analyzed within the CD45−DAPI− population.
4
Isolation and RNA-Seq Analysis of Leukemia Cells
5
Cell Sorting for B2M and HA Expression
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HEK293FT ΔB2M cells were stained with αB2M-FITC antibody (BioLegend) at a dilution of 1:100 for 45 min at 4 °C. Subsequently, cells were washed twice and sorted for B2M negative cells on a Sony SH800 sorter. Jurkat E6 + surface-HA cells were stained with aHA-PB450 (1:100) for 45 min at 4 °C, washed twice with Flow buffer (PBS+2% FBS+5 mM EDTA) and sorted into four bins of different expression levels on a Sony MA900 sorter.
6
Cloning and Expression of H3.3 Variants
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Synthetic genes of H3F3B-mScarlet (H3.3-mScarlet) and H3F3B(K9M)-mScarlet (H3.3K9M-mScarlet) were purchased from IDT provided in pUCIDT cloning vector. Genes were then PCR amplified and TA cloned into pCR8/GW and sequence confirmed. Constructs were then subcloned into pEF-DEST51 backbone using Gateway recombination (Invitrogen). Transfection of A375 cells were performed using Lipofectamine 3000 Reagent (ThermoFisher Scientific). Transfected cells were grown under selection (4 µg/ml blasticidin) 24 h post-transfection for 14 days. A375 cells were sorted using either a BD Influx or Sony MA900 sorter.
7
Intracellular Protein Detection by Flow Cytometry
8
WNK3 Knockdown in T Cell Subsets
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For WNK3 knockdown in human CD4+ and CD8+ T cells, live CD4+ and CD8+ T cells were sorted from human PBMCs using an MA900 sorter (SONY, San Jose, CA, USA) and stimulated with plate-bound anti-human CD3 (#BE0001-2, BioXCell, Lebanon, NH, USA)/anti-human CD28 (#BE0248, BioXCell) and 50 IU IL-2 for 24 h. These cells were spin-infected (900 × g, 90 min, 37 °C) with lentiviral supernatant (shNC and shWNK3). Four days after lentiviral infection, cells were selected with 1 μg/ml puromycin for 7 days. Then, the cells were stimulated with Dynabeads Human T-Activator CD3/CD28 (Gibco) for 36 h. BD GolgiStop (#554724, BD Biosciences) was added for the last 6 h of stimulation. After stimulation, the cells were washed, and surface molecules were stained with anti-CD4 (OKT4, BioLegend) and anti-CD8 (SK1, BioLegend). Cells were then fixed with eBioscience/Invitrogen Intracellular (IC) Fixation Buffer (#00-8222-49, Invitrogen), washed with 1x Permeabilization Buffer (#00-8333-56, Invitrogen) and stained with antibodies for anti-perforin (B-D48, BioLegend) and anti-granzyme B (QA16A02, BioLegend).
9
Lentivirus Transduction of CHO Cells
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CHO cells were routinely cultured in 10-cm tissue culture grade plates using high-glucose DMEM supplemented with 10 % fetal bovine serum (R&D systems, Bio-Techne), 110 μg/mL sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), and housed in a humidified incubator at 37 °C and 5 % CO2. In preparation for lentiviral transduction, CHO cells were grown to 70–80 % confluency in six-well plates, aspirated to remove spent media, and treated with purified viral particles resuspended in 0.8 mL DMEM containing 8 μg/mL polybrene (Thermo Fisher Scientific). The cells were spinfected by centrifuging the six-well plates at 1050 × g for 90 min at 30 °C and the returned to the 37 °C incubator for another 48 h. Stably transduced cells were selected by FACS using a Sony MA900 sorter and stored as cryo-stocks in the vapor phase of liquid nitrogen.
10
Isolation of Fibroadipogenic Progenitors
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PDGFR∝Cre-Rosa26Tdtomato mice (3 months old) were sacrificed with CO2, and hindlimb muscles were harvested and digested as described above. After filtration and centrifugation, cell suspensions were then rinsed with 4 ml of FACS buffer (2% of heat inactivated FBS in HBSS). The samples were then divided into tubes considering the controls (unstained, single colors and fluorescence minus one). Cells were then centrifuged at 350×g for 5 minutes at 4°C and supernatant was discarded. Samples were incubated with corresponding antibodies diluted in FACS media (CD45- 2.5 μg/ml, CD31- 5 μg/ml and SCa-1- 0.5 μg/ml) for 30 minutes on a shaker plate. Then, 2–3 ml of FACS media was added to each tube and centrifuged (350×g minutes for 5 minutes at 4°C), supernatant was removed, and samples resuspended in FACS media. DAPI (1 μg/ml) was then added and samples were filtered through a 30 μm cell strainer and loaded into the Sony MA900 sorter. The gating strategy used to isolate Sca-1+ cells is shown on Supp. Fig. 1C –F . The negative (haematopoietic-CD45+ and endothelial-CD31+) and positive surface markers (stem cell antigen 1 or Sca-1+) were used to isolate FAPs,[64 (link)] and FACS-isolated cells were confirmed to be TdTomato+.
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