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96 well autosampler

Manufactured by Thermo Fisher Scientific
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The 96-well autosampler is a laboratory instrument designed to automate the process of sample introduction for analytical instruments. It features a 96-well microplate format, allowing for the processing of multiple samples in a single run. The core function of this autosampler is to accurately and reliably transfer samples from the microplate to the analytical instrument, such as a chromatograph or spectrometer, for analysis.

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Lab products found in correlation

Attune nxt flow cytometer, by Thermo Fisher Scientific (9 mentions) Ghost dye red 780, by Cytek Biosciences (5 mentions) Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (4 mentions) Opti mem, by Thermo Fisher Scientific (4 mentions) Se buffer, by Lonza (2 mentions) Se sf sg buffer, by Lonza (2 mentions) Bfp to gfp reporter, by Addgene (2 mentions) 4d nucleofector, by Lonza (2 mentions) Anti human cd4 percp, by Cytek Biosciences (2 mentions) Anti human cd25 bv421, by BioLegend (2 mentions) Cd4 fitc, by BioLegend (2 mentions) B2m fitc, by BioLegend (2 mentions) B2m pe, by BioLegend (2 mentions) B2m apc, by BioLegend (2 mentions) Tcra b bv421, by BioLegend (2 mentions) Ghost dye violet 450, by Cytek Biosciences (2 mentions) Cd8 pe cy7, by BD (2 mentions) Rho123, by Merck Group (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Lsrfortessa x 50 flow cytometer, by BD (1 mentions)

10 protocols using 96 well autosampler

1

Cas9-VLP-Mediated Gene Editing in BFP Cells

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Cas9-VLPs targeting BFP were produced as previously described (see Table S1 for guide sequence). Cas9-VLPs were mixed with a single-stranded DNA template (IDT) in either DPBS (Thermo Fisher Scientific), Opti-MEM (Thermo Fisher Scientific), or SE/SF/SG buffer (Lonza). Unless otherwise noted, SE buffer (Lonza) with pulse code CM-150 was utilized. The mixture was electroporated using a 4D-nucleofector (Lonza) before immediately adding to 293T cells stably expressing a BFP-to-GFP reporter (Addgene plasmid #71825). A three nucleotide conversion within the BFP gene results in GFP expression. Cells were analyzed for loss of BFP (non-homologous end joining) and gain of GFP (homology-directed repair) expression after 5–7 days on a Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific).
Hamilton J.R., Tsuchida C.A., Nguyen D.N., Shy B.R., McGarrigle E.R., Sandoval Espinoza C.R., Carr D., Blaeschke F., Marson A, & Doudna J.A. (2021). Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell reports, 35(9), 109207.
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2

MTT Assay for Cytotoxicity Assessment

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3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Rho123 and Histopaque-1077 were purchased from Sigma Aldrich, (Sigma-Aldrich Co., St Louis, MO). Doxorubicin hydrochloride (99.8%, Synbias Pharma Ltd.) was obtained from Nanox Release Technology (Buenos Aires, Argentina) and a fresh solution was prepared by dissolving it in bi-distilled water. Verapamil hydrochloride 98.0%, provided by Parafarm (Buenos Aires, Argentina), was dissolved in ethanol. Measurement of the fluorescence was performed with a Life Technologies Attune-NxT flow cytometer with 96-well autosampler (Thermo Fisher Scientific, USA).
Laiolo J., Lanza P.A., Parravicini O., Barbieri C., Insuasty D., Cobo J., Vera D.M., Enriz R.D, & Carpinella M.C. (2021). Structure activity relationships and the binding mode of quinolinone-pyrimidine hybrids as reversal agents of multidrug resistance mediated by P-gp. Scientific Reports, 11, 16856.
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3

Isolation and Flow Cytometry of Primary Human T Cells

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Primary human T cells were collected 5 days after electroporations, resuspended in FACS buffer, and stained with Ghost Dye Red 780 (Tonbo), anti-human CD4-PerCP (Tonbo, Cat #67-0047-T500) and anti-human CD25-BV421 (Biolegend, Cat #302630). All primary human T-cell gating strategies included singlet gating, live–dead differentiation, and CD4 and CD8 T-cell differentiation and excluded subcellular debris. All quantified data for experiments using primary human T cells were collected from gated CD4+ T cells only.
Cells were resuspended in FACS buffer (1% bovine serum albumin in phosphate-buffered saline) and analyzed by flow cytometry for mNeonGreen+ cells 7 days post-transfection. Flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific), and data analysis was performed using the FlowJo v10 software.
Lin-Shiao E., Pfeifer W.G., Shy B.R., Saffari Doost M., Chen E., Vykunta V.S., Hamilton J.R., Stahl E.C., Lopez D.M., Sandoval Espinoza C.R., Deyanov A.E., Lew R.J., Poirer M.G., Marson A., Castro C.E, & Doudna J.A. (2022). CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells. Nucleic Acids Research, 50(3), 1256-1268.
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4

Cas9-VLP-Mediated Gene Editing in BFP Cells

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Cas9-VLPs targeting BFP were produced as previously described (see Table S1 for guide sequence). Cas9-VLPs were mixed with a single-stranded DNA template (IDT) in either DPBS (Thermo Fisher Scientific), Opti-MEM (Thermo Fisher Scientific), or SE/SF/SG buffer (Lonza). Unless otherwise noted, SE buffer (Lonza) with pulse code CM-150 was utilized. The mixture was electroporated using a 4D-nucleofector (Lonza) before immediately adding to 293T cells stably expressing a BFP-to-GFP reporter (Addgene plasmid #71825). A three nucleotide conversion within the BFP gene results in GFP expression. Cells were analyzed for loss of BFP (non-homologous end joining) and gain of GFP (homology-directed repair) expression after 5–7 days on a Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific).
Hamilton J.R., Tsuchida C.A., Nguyen D.N., Shy B.R., McGarrigle E.R., Sandoval Espinoza C.R., Carr D., Blaeschke F., Marson A, & Doudna J.A. (2021). Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell reports, 35(9), 109207.
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5

Multiparametric Flow Cytometry Analysis

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All flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific). Cells were resuspended in FACS buffer (1%–2% BSA in PBS) and stained with the surface marker-targeting antibodies: B2M-FITC (Biolegend), B2M-PE (Biolegend), B2M-APC (Biolegend), CD4-FITC (Biolegend), CD8-PeCy7 (BD Biosciences), and TCRa/b-BV421 (Biolegend) and live/dead stains Ghost Dye red 780 (Tonbo) or Ghost Dye violet 450 (Tonbo), prior to analyzing. All analysis was done using the FlowJo v10 software. The gating strategy for flow cytometry can be seen in Figures S5, S6, and S9.
Hamilton J.R., Tsuchida C.A., Nguyen D.N., Shy B.R., McGarrigle E.R., Sandoval Espinoza C.R., Carr D., Blaeschke F., Marson A, & Doudna J.A. (2021). Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell reports, 35(9), 109207.
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6

Flow Cytometry Protocol for Cell Quantification

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Flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific) or on an LSRFortessa X-50 flow cytometer (BD Biosciences). Cells were resuspended in FACS buffer (phosphate-buffered saline (PBS), 2% FBS and 1 mM EDTA) and stained with live–dead stain and surface marker-targeting antibodies (Supplementary Table 3) according to manufacturer-provided instructions. Sampling was at defined volumes (60 µl per well) to quantify cell counts. Cytometry data were processed and analysed using FlowJo software (BD Biosciences).
Foss D.V., Muldoon J.J., Nguyen D.N., Carr D., Sahu S.U., Hunsinger J.M., Wyman S.K., Krishnappa N., Mendonsa R., Schanzer E.V., Shy B.R., Vykunta V.S., Allain V., Li Z., Marson A., Eyquem J, & Wilson R.C. (2023). Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes. Nature Biomedical Engineering, 7(5), 647-660.
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7

Flow Cytometry Protocol for Functional Analysis

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All flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (ThermoFisher Scientific). Unless otherwise indicated, cells were collected 3–5 days postelectroporation, resuspended in fluorescence-activated cell sorting (FACS) buffer (1–2% BSA in PBS) and stained with Ghost Dye red 780 (Tonbo) and the indicated cell-surface and intracellular markers (see Supplementary Table 1 for antibodies). To obtain comparable live cell counts between conditions, events were recorded from an equivalent fixed volume for all samples. For intracellular staining, cells were stained for surface markers and then prepared for intracellular staining using True-Nuclear Transcription Factor staining kits (Biolegend). For experiments demonstrating stimulation response, cells were reactivated 24 h before analysis using ImmunoCult Human CD3/CD28/CD2 T Cell Activation reagent (StemCell). Analysis was done using FlowJo v.10 software. All gating strategies included exclusion of subcellular debris, singlet gating and live:dead stain. Final graphs were produced with Prism (GraphPad), and figures were compiled with Illustrator (Adobe).
Shy B.R., Vykunta V.S., Ha A., Talbot A., Roth T.L., Nguyen D.N., Pfeifer W.G., Chen Y.Y., Blaeschke F., Shifrut E., Vedova S., Mamedov M.R., Chung J.Y., Li H., Yu R., Wu D., Wolf J., Martin T.G., Castro C.E., Ye L., Esensten J.H., Eyquem J, & Marson A. (2022). High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nature biotechnology, 41(4), 521-531.
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8

Multiparametric Flow Cytometry Analysis

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All flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific). Cells were resuspended in FACS buffer (1%–2% BSA in PBS) and stained with the surface marker-targeting antibodies: B2M-FITC (Biolegend), B2M-PE (Biolegend), B2M-APC (Biolegend), CD4-FITC (Biolegend), CD8-PeCy7 (BD Biosciences), and TCRa/b-BV421 (Biolegend) and live/dead stains Ghost Dye red 780 (Tonbo) or Ghost Dye violet 450 (Tonbo), prior to analyzing. All analysis was done using the FlowJo v10 software. The gating strategy for flow cytometry can be seen in Figures S5, S6, and S9.
Hamilton J.R., Tsuchida C.A., Nguyen D.N., Shy B.R., McGarrigle E.R., Sandoval Espinoza C.R., Carr D., Blaeschke F., Marson A, & Doudna J.A. (2021). Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell reports, 35(9), 109207.
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9

Quantifying DNA Repair Pathways

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Cells were resuspended in FACS buffer (1% BSA in PBS) and analyzed by flow cytometry for BFP−/GFP− cells (end-joining pathways) and BFP−/GFP+ cells (HDR pathway) 7 dpt. Flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific). Data analysis was performed using FlowJo v10 software.
Chen E., Lin-Shiao E., Trinidad M., Saffari Doost M., Colognori D, & Doudna J.A. (2022). Decorating chromatin for enhanced genome editing using CRISPR-Cas9. Proceedings of the National Academy of Sciences of the United States of America, 119(49), e2204259119.
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10

Primary human T cell isolation and analysis

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Primary human T cells were collected 5 days after electroporations, resuspended in FACS buffer, and stained with GhostDye red 780 (Tonbo), anti-human CD4-PerCP (Tonbo, Cat #67-0047-T500), and anti-human CD25-BV421 (Biolegend, Cat #302630).
All primary human T cell gating strategies included singlet gating, live:dead differentiation, and CD4 and CD8 T cell differentiation and excluded subcellular debris.
All quantified data for experiments using primary human T cells were collected from gated CD4+ T cells only.
Cells were resuspended in FACS buffer (1% BSA in PBS) and analyzed by flow cytometry for mNeonGreen positive cells 7 days post-transfection. Flow cytometry was performed on an Attune NxT flow cytometer with a 96-well autosampler (Thermo Fisher Scientific), and data analysis was performed using the FlowJo v10 software.
Lin-Shiao E., Pfeifer W., Shy B.R., Doost M.S., Chen E., Vykunta V.S., Hamilton J., Stahl E.C., Lopez D.M., Espinoza C.R.S., Dejanov A.E., Lew R.J., Poirer M.G., Marson A., Castro C.E., & Doudna J.A. (2021). CRISPR-Cas9 mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.
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