To generate the STUB1 knockout line STUB1(−/−), we targeted exons 2 and 3 of STUB1 in a dual-cleavage approach to enhance the efficiency of knockout generation. CRISPR-RNAs (crRNAs) (Integrated DNA Technologies) were designed using the CRISPOR web tool (Haeussler et al., 2016 (link)). The iPSCs of control line CO5 were nucleofected with two crRNA-ATTO550-tracrRNA (Integrated DNA Technologies) ribonucleoprotein complexes with Cas9 protein (Integrated DNA Technologies), followed by fluorescence-activated cell sorting of ATTO550+ cells with a Sony Cell Sorter SH800Z, single cell seeding and manual picking and expansion of clones. Homozygous or heterozygous knockout was validated by PCR analysis and Sanger sequencing. The top five exonic off-target effects, as predicted by CRISPOR for each cleavage site, were excluded by Sanger sequencing in the isogenic control, as well as the generated clones. For detailed experimental setup and characterization of STUB1(−/−) see Schuster et al. (2019) (link).
Sh800z cell sorter
Manufactured by Sony
Sourced in Japan, United States, United Kingdom
The SH800Z cell sorter is a laboratory instrument designed for the analysis and sorting of cells. It utilizes flow cytometry technology to precisely identify and separate different cell populations based on their physical and fluorescent properties.
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Lab products found in correlation
Facscalibur, by BD (4 mentions)
Anti cd34 pe clone qbend 10, by Thermo Fisher Scientific (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Irf4 3e4, by BD (2 mentions)
Aqua dead cell staining kit, by Thermo Fisher Scientific (2 mentions)
Fc block 2.4g2, by BD (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Fortessa, by BD (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Cas9 protein, by Integrated DNA Technologies (1 mentions)
Syto bc, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti mouse iga, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Transit 2020 transfection reagent, by Mirus Bio (1 mentions)
Annexin 5 allophycocyanin apc, by BioLegend (1 mentions)
40 protocols using sh800z cell sorter
1
Generation of STUB1 Knockout Cell Line
2
Flow Cytometry Analysis of TRA-1-60 Expression
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Flow cytometry was performed as described previously [33 (link)]. The cells were resuspended at approximately 1 × 106 cells/mL in MACS buffer and incubated with anti–TRA-1-60 antibodies (1:300 dilution; clone TRA-1-60, Merck Millipore) for 1 h at 4 °C. Normal mouse IgM (Merck Millipore) was used as an isotype control. The cells were rinsed with MACS buffer and then incubated with Alexa Fluor 488 goat anti-mouse IgM (1:300 dilution; Thermo Fisher Scientific). After further rinsing, cells were stained with propidium iodide (PI) (Thermo Fisher Scientific), and 20,000 cells were analysed using a Cell Sorter SH800Z (Sony Corporation, Tokyo, Japan). The data were analysed with FlowJo software (BD Biosciences).
3
Isolation and Sequencing of IgA-Coated Bacteria
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IgA-coated bacteria were isolated and sequenced as previously described19 (link),20 (link). In brief, frozen fecal samples were thoroughly homogenized in PBS to a final concentration of 100 mg/mL. Fecal suspensions were filtered through a 40-μm sterile nylon mesh, then centrifuged at 50 × g, for 15 min at 4 °C. 100 μL of supernatant was then washed twice with 1 mL of staining buffer (PBS containing 1% (w/v) BSA) and centrifuged at 50 × g, for 15 min at 4 °C. Resulting bacterial pellets were resuspended in 100 μl blocking buffer (staining buffer containing 20% Normal Rat Serum) and incubated for 20 min on ice before being stained with 100 μl of staining buffer containing PE-conjugated Anti-Mouse IgA (1:12.5; eBioscience, 12–4204–82) for 30 min on ice. Following three washes with staining buffer, pellets were resuspended in 200 μL of 0.9%NaCl/0.1 m HEPES buffer (pH 7.2) containing a 1:4000 dilution of SytoBC (Invitrogen, S34855). Data acquisition was performed on a Sony Cell Sorter SH800Z. Samples were gated on appropriate SSC-A/FSC-A gates prior to being selected for SytoBC+ events. For each sample, 100,000 events were collected from the IgA− and IgA+ population into sterile tubes. Each fraction was stored at − 20 °C prior to DNA extraction and sequencing of bacterial 16 S rRNA genes, as described above.
4
Fluorescence-Activated Cell Sorting
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Cells were sorted by fluorescence on a Sony Cell Sorter SH800Z (100 μm sorting chip) equipped with 488 nm excitation laser and 525/50 emission filter (eGFP), and 561 nm excitation laser and 600/60 emission filter (mCherry). Data were analyzed using Sony Cell Sorter Software v2.1.5.
5
Spleen Cell Sorting for Follicular B Cells
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Spleen cells for sorting were prepared from tissue and surface-stained same way as in flow cytometry analysis. For follicular B cell sorting, total splenic B cells were first enriched prior to sorting. Single-cell suspensions, FMO controls, and single-color stain compensation samples were resuspended in sterile HBSS buffer with 10 mM HEPES, 1 mM EDTA, and 2% FBS at appropriate concentrations and filtered immediately prior to sorting. Cells were sorted into RPMI media with 10% FBS and kept on ice until use. Two sorters were utilized: 5-laser (488nm, 406nm, 592nm, 640nm, and 355nm) FACSAria-SORP cell sorter with PMT-forward scatter option; Sony cell sorter SH-800Z with 4 laser (405nm, 488nm, 561nm, and 633nm), 6-color configuration and 70 μM sorting chips.
6
Flow Cytometry Analysis of PBMC
7
CRISPR Cas9 Knockdown of Ninj1
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CRISPR Cas9 All-in-one lentiviral expression vectors targeting Ninj1 were purchased from transOMIC Technologies inc. (Huntsville, AL, USA). MLE-12 and Raw264.7 cells were transfected with the CRISPR Cas9 expression vector using TransIT®−2020 Transfection Reagent (Mirus Bio LLC), following manufacturer’s instruction. The transfected cells were sorted by tRFP, using SONY cell sorter SH800Z (SONY, Tokyo, Japan). The sorted cells were plated on 96-well plate for single cell colony selection. Ninj1 expression was evaluated by performing western blot analysis. Ninj1-expressing cells were referred to as WT and Cas9-drived Ninj1-deficient cells as Ninj1 KO.
8
Enriching Live Single Epithelial Cells
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Single cells were washed with dPBS + Y-27632, resuspended in Advanced Dulbecco’s modified Eagle medium/F12 + 1% bovine serum albumin + Y-27632, and then stained with AnnexinV-Allophycocyanin (APC) (1:100) and 1 TotalSeq Anti-Human Hashtag Antibody (1:100, Biolegend, San Diego, CA) per region to track all 6 regions with a single library preparation.11 (link) Cells were washed and resuspended in Advanced Dulbecco’s modified Eagle medium + 1% bovine serum albumin + Y-27632 for sorting on a Sony Cell Sorter SH800Z (Sony, Tokyo, Japan) to enrich for live single epithelial cells (Figure 18 ). There were 25,000 cells that passed Annexin V live/dead parameters and were fluorescence-activated cell sorter isolated for each region, then different cell hashing antibodies were added to cells from each of the 6 regions before pooling for library preparation. An additional live/dead analysis was performed after pooling and approximately 10,000 cells from the pooled population were loaded onto the Chromium Next GEM Single Cell 3’ GEM, Library and Gel Bead Kit v3.1 (PN-100012, 10x Genomics, Pleasanton, CA) for complementary DNA library preparation Sequencing was performed on an Illumina NextSeq 500 (Illumina, San Diego, CA).![]()
9
Primate EB FACS Isolation Protocol
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FACS was performed as previously described with some minor modifications (Kobayashi et al, 2017 (link)). Briefly, primate EBs were harvested on day 4, washed with PBS, and incubated in 0.25% trypsin/EDTA in a thermomixer at 850 rpm at 37°C for 9–13 min. The reaction was stopped with 3% FBS in PBS, and the cell suspension was pipetted until it was dissociated into single cells and then passed through the strainer. The cells were centrifuged, and the cell pellet was resuspended in 3% FBS in PBS. Then, the dissociated cells were stained for CXCR4-APC (306510; BioLegend) and INTα6-BV421 (313624; BioLegend) for 1 h in the dark on ice. Afterwards, the cells were washed in 3% FBS in PBS and subjected to FACS using SONY Cell Sorter SH800Z.
10
Ninj1 Regulates Cell-Cell Interactions
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WT or Ninj1 KO MLE-12 cells with or without BLM (50 µg/ml) treatment for 12 hours and CFSE-stained WT or Ninj1 KO Raw264.7 cells were co-cultured for 30 min, and the number of Raw264.7 cells bound to MLE-12 cells was assessed using the BD FACSCalibur (BD Bioscience). For RNA and protein analysis, after co-culture of WT or Ninj1 KO MLE-12 and CFSE-stained WT or Ninj1 KO Raw264.7 for 6 hours, Raw264.7 cells were sorted using the SONY cell sorter SH800Z (SONY, Tokyo, Japan), and RNA and protein were isolated from the sorted Raw264.7 cells. To collect conditioned media (CM), the media was changed to serum-free medium after 6 hours of co-culture and CM was collected 12 hours after media change.
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