Mouse cd8 t cell enrichment kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The Mouse CD8 T cell enrichment kit is a laboratory tool designed to isolate CD8 T cells from mouse samples. It utilizes magnetic bead-based separation technology to selectively enrich for the CD8 T cell population.

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Lab products found in correlation

100 μm nylon cell strainer, by BD (2 mentions) Anti cd8 clone 53 6, by Thermo Fisher Scientific (1 mentions) 100 m nylon cell strainer, by BD (1 mentions) Facsaria fusion instrument, by BD (1 mentions) Anti biotin microbead, by Miltenyi Biotec (1 mentions) Klrg1 2f1, by Thermo Fisher Scientific (1 mentions) Cd45.1 a20, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Roche (1 mentions) Collagenase d, by Roche (1 mentions) 70 μm nylon cell strainer, by BD (1 mentions) Anti cd8a apc clone 53 6, by BioLegend (1 mentions) Facsfusion, by BD (1 mentions) Cd4 fitc clone rm4 4, by BioLegend (1 mentions)

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CD8+ T cells (21 citations)

4 protocols using mouse cd8 t cell enrichment kit

Isolation of Chronic P14 T Cells

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Single-cell splenocyte suspensions were obtained by mashing total spleens through a 100-µm nylon cell strainer (BD), and red blood cells were lysed with a hypotonic ACK buffer. Transgenic CD8⁺ T cells were isolated using the mouse CD8 T cell enrichment kit (Miltenyi Biotech). 2–5 × 10³ CD45.1⁺ naive congenic P14 T cells were transferred into naive CD45.2⁺ C57BL/6 mice. P14 T cells were reisolated from infected mice by staining total splenocytes with anti-CD8 (clone 53-6.7; eBioscience), CD45.1 (purified hybridoma supernatant; self-conjugated; clone A20), and CD127 (clone eBioSB/199; eBioscience). CD8⁺CD45.1⁺ or CD8⁺CD45.1⁺CD127⁺ cells were isolated by flow cytometry–assisted cell sorting. The purity of sorted cells was >99%. In all cases, we confirmed that T cells isolated from pure clone-13–infected mice exhibit the chronic phenotype.

Utzschneider D.T., Alfei F., Roelli P., Barras D., Chennupati V., Darbre S., Delorenzi M., Pinschewer D.D, & Zehn D. (2016). High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival. The Journal of Experimental Medicine, 213(9), 1819-1834.

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Isolation and Transfer of Memory CD8+ T Cells

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Single-cell suspensions were obtained by mincing spleens with a scalpel and then by mashing them through a 100 μm nylon cell strainer (BD Falcon). Red blood cells were lysed with hypotonic ammonium-chloride-potassium (ACK) lysis buffer. The mouse CD8⁺ T cell-enrichment kit (Miltenyi Biotech) was used for CD8 T cell-isolation. Memory OT-I T cells were generated by transferring low numbers of naive OT-I T cells into CD45.1 congenic host mice and by infecting the hosts with 1000 CFU of Lm-N4. Memory cells were re-isolated via staining and positive selection of live cells in 2% FCS RPMI medium (Sigma Aldrich). First, cells were stained with fluorescein isothiocyanate (FITC)-KLRG1, or phycoerythrin (PE)-CD127, and biotin-conjugated CD45.2 antibodies, and then enriched via MACS separation using anti-biotin MicroBeads (Miltenyi Biotech), in accordance with the manufacturer’s instructions. Afterward, cells were sorted for KLRG1⁺ or CD127⁺ populations using a FACS Aria Fusion instrument (BD) and then transferred into new CD45.1/1-congenic C57BL/6J host mice.

Flosbach M., Oberle S.G., Scherer S., Zecha J., von Hoesslin M., Wiede F., Chennupati V., Cullen J.G., List M., Pauling J.K., Baumbach J., Kuster B., Tiganis T, & Zehn D. (2020). PTPN2 Deficiency Enhances Programmed T Cell Expansion and Survival Capacity of Activated T Cells. Cell Reports, 32(4), 107957.

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Isolation of Immune Cell Subsets

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Single-cell splenocyte suspensions were obtained by mashing total spleens through a 70-μm nylon cell strainer (BD), and red blood cells were lysed with a hypotonic ammonium chloride-potassium bicarbonate (ACK) buffer. Lymph nodes were homogenized by teasing between frosted glass slides followed by filtration through nylon cell strainer. IEL cells were isolated as previously described (Lefrancois and Lycke, 2001 ). Liver was homogenized and cells recovered after Percoll separation. Lungs and kidneys were cut in small pieces and digested in 1 mg/mL collagenase D and 0.1 mg/mL DNase I (Roche) for 2 hr at 37°C. Tissues were then homogenized and passed through a nylon strainer followed by an ACK lysis.
Transgenic naive iFx1^f/f- and WT-P14 were isolated using the mouse CD8⁺ T cell enrichment kit (Miltenyi Biotech), and 10³–10⁵ P14 were transferred into naive CD45.1⁺ C57BL/6 mice. P14 cells were re-isolated from infected mice by staining total splenocytes in 10% FBS RPMI media with anti-CD8α (53-6.7), CD45.1 (A20), CD45.2 (104), and KLRG1 (2F1; all from ThermoFisher).

Utzschneider D.T., Delpoux A., Wieland D., Huang X., Lai C.Y., Hofmann M., Thimme R, & Hedrick S.M. (2018). Active Maintenance of T Cell Memory in Acute and Chronic Viral Infection Depends on Continuous Expression of FOXO1. Cell reports, 22(13), 3454-3467.

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Isolation and Sorting of Naive Transgenic CD8 T Cells

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Single-cell splenocyte suspensions were obtained by mashing total spleens through a 100-μm nylon cell strainer (BD Falcon) and lysing red blood cells with a hypotonic ACK buffer. Naive transgenic P14 CD8 T cells were isolated using the mouse CD8+ T cell Enrichment Kit (Miltenyi Biotech, Bergisch-Gladbach, Germany). Surface staining was performed for 40 min at 4 °C in supplemented Dulbecco’s modified Eagle’s medium (DMEM) media (Gibco, ThermoFisher Scientific) using the following antibodies: anti-CD8a-APC (clone 53-6.7; dilution 1:400; Biolegend), CD4-FITC (clone RM4-4; dilution 1:400; Biolegend), and TCR V alpha 2-PE (clone B20.1; dilution 1:400; eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle, standard operation settings, single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq), which was subsequently spun down, snap-frozen on dry ice, and stored at −80 °C until use. All data were analyzed using FlowJo (TreeStar).

Kanev K., Roelli P., Wu M., Wurmser C., Delorenzi M., Pfaffl M.W, & Zehn D. (2021). Tailoring the resolution of single-cell RNA sequencing for primary cytotoxic T cells. Nature Communications, 12, 569.

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