Purified CD4+ T cells were infected with HIVGKO as described above. On day 5 post-infection, cells were labeled with live/dead (VIVID) stain and GFP negative, latent cell-enriched gated population was sorted in purity mode using Sony SH800 sorter. The sorting gate contained ~10% of latent cells by fluorescent reporter expression (mKO2+GFP-) and the rest were uninfected (GFP-). Cells were collected in complete medium (R10), centrifuged, and resuspended in fresh medium and divided equally into 4 wells of a 96 well plate with approximately 104 cells/well in 150ul of R10. The cells rested overnight in presence or absence of physiologically relevant concentrations of (0.1, 1, and 10 µM) baricitinib (Selleck chemicals) before addition of LRA (1 µg/mL each of anti-CD3 and anti-CD28). Two days after LRA treatment, cells were stained for live/dead (VIVID) and acquired on Sony SH800 sorter to assess GFP and mKO2 expression for at least 25,000 cells (events). Flow cytometric data were analyzed using FlowJo version 10.7.1.
Sh800 sorter
Manufactured by Sony
Sourced in Japan
The SH800 is a cell sorter designed for high-performance cell sorting and analysis. It features a compact design, precise fluidics, and advanced optics for efficient cell separation and data collection.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Single cell sort mask, by Sony (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Rpmi 1640 glutamax, by Thermo Fisher Scientific (2 mentions)
4k54 diet, by Land O'Lakes (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Dnase 1, by Roche (2 mentions)
Collagenase 1, by Worthington (2 mentions)
Dead cell removal kit, by STEMCELL (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Cd3 percp cy5, by Thermo Fisher Scientific (2 mentions)
Cd138 pe 44f9, by Miltenyi Biotec (2 mentions)
Slamf7 apc 162, by BioLegend (2 mentions)
Bcma pe cy7 19f2, by BioLegend (2 mentions)
Cd14 apc cy7 mop9, by BD (2 mentions)
Sytox blue, by Thermo Fisher Scientific (2 mentions)
Draq7, by Cell Signaling Technology (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Facsaria 2 machine, by BD (2 mentions)
47 protocols using sh800 sorter
1
HIV Latency Reactivation Assay
2
SynNotch T Cell Cytotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA-MB-231 target cell lines were
generated through lentiviral transduction and subsequent sorting with
a SONY SH800 sorter. For cytotoxicity assays, 2.5 × 104 CD19 positive and 2.5 × 104 CD19 negative MDA-MB-231
cells were cocultured with 2.5 × 105 CD19-SynNotch
monobody-CAR T cells in 150 μL of RPMI for 24 h. Bioluminescence
measurements were taken using the Dual Glo Luciferase Assay kit (Promega,
model E2920). Cytotoxicity was measured by calculating the percent
difference in luminescence of SynNotch T cells versus that of target
cancer cells only.
generated through lentiviral transduction and subsequent sorting with
a SONY SH800 sorter. For cytotoxicity assays, 2.5 × 104 CD19 positive and 2.5 × 104 CD19 negative MDA-MB-231
cells were cocultured with 2.5 × 105 CD19-SynNotch
monobody-CAR T cells in 150 μL of RPMI for 24 h. Bioluminescence
measurements were taken using the Dual Glo Luciferase Assay kit (Promega,
model E2920). Cytotoxicity was measured by calculating the percent
difference in luminescence of SynNotch T cells versus that of target
cancer cells only.
3
Optimizing Single Cell Sorting
4
Single-Cell Transcriptomics Using Fluidigm
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluidigm was performed per manufacturer’s instructions. In brief, frozen cells were thawed as and single cells were sorted into 96 wp containing lysis buffer using a SONY SH800 sorter. After reverse transcription was performed (using 10× Superscript VILO Enzyme Mix, per manufacturer’s protocol (ThermoFisher), specific target amplification was performed using the primer pool provided by Fluidigm (employing Fluidigm custom primers) and the TaqMan PreAmp Master Mix (ThermoFisher). After the IFC plate was primed, SsoFast EvaGreen SuperMix (BioRad) and the diluted specific amplification product were loaded onto the IFC plate and run on the BioMark HD. Data was processed using the R library “fluidigmSC” and individual cell gene expression was graphed using GraphPad Prism.
5
Macrophage Isolation from Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Another filter step followed and the centrifuged cells were resuspended in 40 µL FACS buffer (PBS containing 2 mM EDTA and 0.5% bovine serum albumin) containing 10 µL of FcR Blocking Reagent (Miltenyi Biotec). The cells were blocked for 10 min at 4 °C. Then, the cells were incubated with CD45 magnetic beads (Miltenyi Biotec) for 15 min at 4 °C and the leukocyte fraction was separated from other cell fractions using AutoMACS (Miltenyi Biotec) with the program ‘possel’. Immune cell-enriched fractions from the lung were stained with Zombie Violet dye (BioLegend) and antibodies against CD45-FITC (HI30, 1:20 dilution, BioLegend) and CD206-PECy5 (15-2, 1:20 dilution, Sony Biotechnology) to isolate macrophages. Cells were analysed using Sony SH800 sorter using the 130 µm sorting chip.
6
Zymosan-Induced Peritoneal Macrophage Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Male C57BL/6 mice were randomly assigned to experimental groups. Mice were injected I.P. with zymosan A (1 mg/ml in PBS, 1 ml per mouse). PKH2-PCL green (0.25 mM; 0.5 ml; Sigma-Aldrich) was injected I.P. at 20, 44, 62, or 68 h, and peritoneal exudates were collected 4 h later. Peritoneal cells were stained with PE- or Brilliant violet-conjugated rat anti-mouse F4/80, PerCP-conjugated rat anti-mouse CD11b, Pacific Blue- or PerCP-conjugated rat anti-mouse Ly6C, PE-conjugated rat anti-mouse CD115, and PE/Cy7-conjugated mouse anti-mouse CX3CR1 (Biolegend) and analyzed by flow cytometry as in Supplemental Figure 1 . F4/80+macrophages were sorted according to PKH2-PCL green signal intensity as in (17 (link)) using the FACSaria III sorter (Beckton-Dickinson) to give distinct F4/80+/PKH2hi and F4/80+/PKH2lo/neg macrophage populations. Ly6CmedF4/80neg and Ly6ChiF4/80lo monocytic cells were sorted using the SH800 sorter (Sony). In some experiments, flow cytometry analysis using the FlowJo software (Treestar) was performed to identify distinct leukocyte populations as detailed in the results section.
7
Optimizing Single Cell Sorting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were sorted using a 130 μm chip. Because the single cell sort mask on the Sony instrument is more lenient and sorts all droplets in the vicinity of detected target events as long as off target events are not nearby, we found calibration of drop delay values unnecessary. Instead the sort recovery and purity of nanovial sorts was evaluated using the four primary sort masks, yield, normal, purity, and single cell. In each condition 200 AlexaFluor™ 488 Streptavidin target nanovial events were sorted from a background of AlexaFluor™ 647 Streptavidin labeled particles. Recovered samples were analyzed using fluorescent microscopy. Note, the Sony SH800 sorter by default reports forward and back scatter data. Generated back scatter data was converted to side scatter upon export and is referred to as such throughout the manuscript.
8
Lentiviral Transduction of TCR Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lentivirus encoding transfection of TCRα and TCRβ chain genes was generated as described above 1 .
Brie y, we cloned either the full-length TCRα or TCRβ chain gene into the pHR lentiviral vector. After lentivirus production, 2 mL of the supernatant containing TCR α and β chains with 1:1 expression was used to infect 1 million SKW3 cells. Following infection, we added 1.5 mL fresh completed RPMI to the mixture. After 2 days, we selected for SKW3 cells with surface-expressed TCRαβ using anti-TCR (Biolegend clone IP26) staining and ow cytometry (Sony SH800 sorter).
Cell culture SKW3 cells transduced with TCR clones 55, 589 and DMF5 were cultured in RPMI 1640 GlutaMAX (Thermo Scienti c) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin, and 100 U/ml streptomycin (Life technology). Prior to performing all experiments, we con rmed that cells had reached the log phase of growth (1.5 million to 2 million cells/mL), as cells in the lag phase may generate insu cient Ca 2+ ux (https://osf.io/xs7zf/).
Brie y, we cloned either the full-length TCRα or TCRβ chain gene into the pHR lentiviral vector. After lentivirus production, 2 mL of the supernatant containing TCR α and β chains with 1:1 expression was used to infect 1 million SKW3 cells. Following infection, we added 1.5 mL fresh completed RPMI to the mixture. After 2 days, we selected for SKW3 cells with surface-expressed TCRαβ using anti-TCR (Biolegend clone IP26) staining and ow cytometry (Sony SH800 sorter).
Cell culture SKW3 cells transduced with TCR clones 55, 589 and DMF5 were cultured in RPMI 1640 GlutaMAX (Thermo Scienti c) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin, and 100 U/ml streptomycin (Life technology). Prior to performing all experiments, we con rmed that cells had reached the log phase of growth (1.5 million to 2 million cells/mL), as cells in the lag phase may generate insu cient Ca 2+ ux (https://osf.io/xs7zf/).
9
Isolation of Cardiomyocytes from Alms1 Gt/Gt Mice
10
Isolation of Cardiomyocytes from Alms1 Gt/Gt Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Alms1Gt/Gt mice (B6.129P2-Alms1Gt(XH152)Byg/Pjn) used in this study were generated from gene-trapped ES cells (MMRC#008633)26 (link),47 (link). Mice were fed ad libitum a 4K54 diet (PMI Nutrition International, St. Louis, MO) and provided an unlimited access to water in a temperature/humidity controlled setting with a 12 hour light/dark cycle at The Jackson Laboratory Research Animal Facility. All mouse protocols used in this study were approved by the JAX institutional Animal Care and Use Committee. Mouse hearts were extracted from mice euthanized by carbon dioxide asphyxiation and allowed to contract in phosphate buffered saline for 5–10 minutes and immediately placed in 4% paraformaldeyde at 4 °C overnight. Subsequently, tissues were embedded in paraffin, sectioned, and immunostained as previously reported48 (link). The aMHC promoter-driven EGFP-IRES-puromycin transgenic mice (alphaMHC-GFP), in which only cardiomyocytes express the green fluorescent protein (GFP) facilitate rapid and efficient isolation of cardiomyocytes23 (link). Hearts from these mice were dissociated using collagense II/TrypLE. GFP+ cardiomyocytes were isolated by Fluorescence Activated Cell Sorting (FACS) using an SH800 sorter (Sony Biotechnology).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!