hMDMs were lifted from the 12-well plate by vigorously washing the cells off the plate with the media. Cold PBS with EDTA (10 mM) was added, and plates were incubated at 4C for 10min, followed by a second vigorous wash, and a PBS wash. The different infection conditions were tagged based on expression of cell surface markers B2M and CD298 using the Cell Hashing method (Stoeckius et al., 2018 (link)) following their protocol (Table S2 ). Eight samples (7 bacteria and an unexposed control) or ten samples (9 Staphylococcus strains and unexposed) from the same donor were pooled into a single cell suspension in equal amounts (Figure S4B ), and ran on the 10X Genomics Chromium Controller with Single Cell 3′ v3 system. The remaining cells were analyzed on a FACS- SONY SH800 to quantify the proportion of infected cells using the fluorescent label of the bacteria inside the cells (Figure S4A ). cDNA amplification and library preparation of the mRNA was processed according to 10X Genomics Single Cell 3′ v3 manufacturer’s instructions. For the hashtag oligos, 1ul of HTO PCR additive primer was added to the 10X cDNA amplification step, and the supernatant from the 0.6X cDNA cleanup was kept and processed according to the Cell Hashing protocol, with 14 PCR cycles and a 1.2X cleanup after the PCR.
Facs sh800
Manufactured by Sony
Sourced in United States, Japan
The FACS SH800 is a cell sorter system designed for automated flow cytometry analysis and sorting. It features a compact and modular design, supporting up to 4 lasers and 14 fluorescence parameters. The system is capable of high-speed cell sorting at rates up to 70,000 cells per second.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Fujifilm (2 mentions)
Siglecf apc, by Thermo Fisher Scientific (2 mentions)
Mhcii apc cy7, by Thermo Fisher Scientific (2 mentions)
Dispase 2, by Roche (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Cd11c pe, by BD (2 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Streptavidin ecd, by Beckman Coulter (1 mentions)
Anti cd49f pe, by Thermo Fisher Scientific (1 mentions)
Anti cd4 percp, by BioLegend (1 mentions)
Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions)
Cell sorter software, by Sony (1 mentions)
7 aad, by BD (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Clone 30 f11, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Ex527, by Selleck Chemicals (1 mentions)
Clone 390, by Thermo Fisher Scientific (1 mentions)
17 protocols using facs sh800
1
Hashed Single-Cell RNA-Seq of Infected Macrophages
2
Hashed Single-Cell RNA-Seq of Infected Macrophages
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hMDMs were lifted from the 12-well plate by vigorously washing the cells off the plate with the media. Cold PBS with EDTA (10 mM) was added, and plates were incubated at 4C for 10min, followed by a second vigorous wash, and a PBS wash. The different infection conditions were tagged based on expression of cell surface markers B2M and CD298 using the Cell Hashing method (Stoeckius et al., 2018 (link)) following their protocol (Table S2 ). Eight samples (7 bacteria and an unexposed control) or ten samples (9 Staphylococcus strains and unexposed) from the same donor were pooled into a single cell suspension in equal amounts (Figure S4B ), and ran on the 10X Genomics Chromium Controller with Single Cell 3′ v3 system. The remaining cells were analyzed on a FACS- SONY SH800 to quantify the proportion of infected cells using the fluorescent label of the bacteria inside the cells (Figure S4A ). cDNA amplification and library preparation of the mRNA was processed according to 10X Genomics Single Cell 3′ v3 manufacturer’s instructions. For the hashtag oligos, 1ul of HTO PCR additive primer was added to the 10X cDNA amplification step, and the supernatant from the 0.6X cDNA cleanup was kept and processed according to the Cell Hashing protocol, with 14 PCR cycles and a 1.2X cleanup after the PCR.
3
Quantifying Gene Conversion and NHEJ Repair
4
Isolation of Mammary Stem Cells
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A dissociated single-cell suspension was labeled with biotinylated CD45, Ter119, CD31, and BP-1 antibodies contained in EasySep Mouse Epithelial Cell Enrichment Cocktail (StemCell Technologies) for 30 min on ice, and after washing, incubated with anti-CD49f-PE (eBioscience, San Diego, CA, USA), anti-CD24-perCP-cy5.5 (eBioscience), streptavidin-ECD (Beckman-Coulter, Brea, CA, USA), and 7-amino-actinomycin D (7-AAD; Calbiochem, San Diego, CA, USA) for 30 min on ice. Cells were suspended in 2% FBS–Hank’s balanced salt solution including DNaseI before sorting. Cell sorting was carried out using fluorescence-activated cell sorting (FACS) SH800 (Sony, Tokyo, Japan). CD45− Ter119− CD31− BP-1− (Lin(−)) 7-AAD− CD49fhigh CD24+ cells were collected to represent the MaSC-enriched fraction.
5
Single-Cell Sorting via FACS
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DEs were loaded and analyzed on the FACS SH800 (Sony) using a standard 408 nm laser configuration and a 130 μm microfluidic chip for sorting. Droplets were first gated on FSC and SSC profile, followed by singlet gating using FSC-H and FSC-A and subsequent gating on APC, FITC or DAPI fluorescence, as indicated. All flow and thresholding parameters are reported in Table 2 . Sorts were completed with single cell purity mode.
6
Single-Cell Sorting via FACS
7
Genomic DNA Extraction and Chimerism Analysis in Rats
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Genomic DNA of chimeric rats was extracted from liver pieces or fluorescent-negative fraction of splenic lymphocytes sorted by fluorescence-activated cell sorting (FACS, SH800; Sony Corp., Tokyo, Japan). For HR#4 rat line, the Sall1 locus was amplified by PCR using F1, R1 and R2 primers (Supplementary Table 2 ). For CRISPR#1 rat line, the Sall1 locus was amplified by PCR using F1, R2 and R3 primers (Supplementary Table 2 ). Splenic lymphocytes were used to determine the fluorescence-based chimerism of each xenogeneic or allogeneic chimeric rat. The fluorescence-positive gates were set using the FACS histograms of the non-chimera samples (negative controls).
8
Stable CD3-Overexpressing 293T Cells
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293T cells (1.1 × 105 cells/well) were seeded into 12-well plates. Next day, the cells were washed with PBS to remove the antibiotics and 1 mL of DMEM supplemented with 10% FBS was added. The CD3 expression vector pEF1α-CD3E-T2A-CD3G-T2A-CD3D-T2A-CD3Z-T2A-EGFP (1 μg) and Lipofectamine 2000 (Thermo Fisher Scientific) were diluted in 50 μL of Opti-MEM (Thermo Fisher Scientific), respectively. After 5 min, the two diluted solutions were combined, incubated at room temperature for 5 min to form DNA-liposome complexes, and transferred into the 293T cells. EGFP+ cells were purified using FACS SH800 (SONY) and maintained to establish a stable transfectant (designated as CD3-OE-293T cells).
9
Isolation of Mouse Regulatory T Cells
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Mice were euthanized by cervical dislocation, then spleen and lymphocytes were dissected and passed through a 70 µM sieve. Erythrocytes were lysed with buffer containing 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.3. Cells were washed twice using Hank’s buffered salt solution (HBSS) containing 2 mM EDTA and 1% penicillin/streptomycin. Cells were then cultured overnight in RPMI164 medium containing 1% penicillin/streptomycin, 50 µM β-mercaptoethanol, and 20 ng/mL IL-2. Next day, Tregs and Tcons were isolated using CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi, #130-091-041) according to manufacturer’s instructions. Alternatively, total CD4 cells were labelled with anti-CD4 PerCP (Biolegend, San Diego, CA, USA, #100431) and isolated using fluorescence-activated cell sorting (FACS-SH800, Sony Biotechnology, CA, USA).
10
Flow Cytometric Analysis of UDCs
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Flow cytometric evaluation of UDCs was performed using fluorescein isothiocyanate-conjugated anti-human CD90 antibody (BioLegend, #328107) and 7-AAD (BD Biosciences, 51-68981E). UDCs were trypsinized and washed with growth medium, centrifuged at 200 × g for 5 min, collected, and suspended in phosphate-buffered saline (PBS) with 2% FBS. The antibody was added to UDC suspension at an optimal concentration (1:200) and incubated for 30 min on ice in the dark. Moreover, the 7-AAD was added to UDC-suspension at an optimal concentration (1:100) and incubated for 10 min before analysis. The cells were centrifuged, resuspended in PBS with 2% FBS, passed through a 70 μm filter, and analyzed using a SONY FACS SH800S (Sony Biotechnology, San Jose, CA, USA) and Cell Sorter Software (Sony).
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