Tumor infiltrated CD8+ T cells were purified with the CD8a (Ly-2) MicroBeads (Miltenyi Biotech), and then were stained with Percp/CY5.5 conjugated anti-CD3, APC conjugated anti-CD8β, BV421 conjugated anti-CD44, CD3+ CD8+ CD44+ or CD3+ CD8+ CD44− cells were sorted by BD FACSAria II. For OTI Trm cell sorting, 1.5 μg FITC conjugated anti-CD45 mice were injected i.v. 5 minutes before mice were scarified. OTI cells were enriched by CD8a (Ly-2) MicroBeads (Miltenyi Biotech), and stained with BV421 conjugated anti-CD8a, APC conjugated anti-CD90.1, and PE conjugated anti-CD90.2. Trm cells were sorted based on (i.v. CD45−) CD8+CD90.1+CD90.2− or (i.v. CD45−) CD8+CD90.1+CD90.2+.
Cd8a ly 2 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
CD8a (Ly-2) MicroBeads are magnetic beads coated with antibodies specific for the CD8a (Ly-2) surface marker. They are used for the isolation and enrichment of CD8+ T cells from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 l3t4 microbeads, by Miltenyi Biotec (7 mentions)
Automacs separator, by Miltenyi Biotec (4 mentions)
Automacs magnetic cell separation system, by Miltenyi Biotec (2 mentions)
Mojosort cd4 isolation kit and magnet, by BioLegend (2 mentions)
Cd4 l3t4, by Miltenyi Biotec (2 mentions)
Red blood cell lysis solution, by Merck Group (1 mentions)
40 μm pore cell strainer, by BD (1 mentions)
Ld separation columns, by Miltenyi Biotec (1 mentions)
Lysis binding buffer, by Thermo Fisher Scientific (1 mentions)
Soluble αcd28 37, by BioXCell (1 mentions)
Quantstudio 3 real time system, by Thermo Fisher Scientific (1 mentions)
Murine il 2, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Cd8a t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Anti ifn γ mab, by BD (1 mentions)
Recombinant mouse ifn γ, by R&D Systems (1 mentions)
35 protocols using cd8a ly 2 microbeads
1
Tumor-Infiltrating CD8+ T Cell Isolation and Sorting
2
Allogeneic Hematopoietic Stem Cell Transplantation
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Recipient male CD45.2 B6 mice were lethally irradiated (11 Gy total body irradiation, split into 2 fractions over a period of 48 h) and reconstituted 4 h after the second dose with 5 × 106 female CD45.1 C57BL/6 BM cells and 2 × 106 CD4 T cells, with 1 × 106 CD8 Thy1.1+ Matahari T cells, administered by intravenous injection through the tail vein. CD4 and CD8 T cells were isolated by magnetic selection of CD4 or CD8 splenocytes using the Miltneyi MACS system (QuadroMACS Separator, LS columns, CD4 [L3T4] MicroBeads, CD8a [Ly-2] MicroBeads; Miltenyi Biotec) for injection of splenic T cells. In some experiments syngeneic controls were use in which female recipients received female BM with Matahari T cells.
3
Splenocyte Isolation and T-Cell Depletion
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Spleens were harvested from immunized mice (n = 9), and single-cell suspensions were prepared by passing the organs through 70-μm-pore cell strainers into RPMI-10. Cells were pelleted by centrifugation (450 × g), resuspended in red blood cell lysis solution (Sigma), and then incubated at room temperature for 10 min. Cells were washed twice by being pelleted (450 × g) and then resuspended in RPMI-10, and then they were passed through a 40-μm-pore cell strainer (Falcon). Prior to treatment for T cell depletion, splenocytes prepared from 3 individual spleens were pooled, resulting in 3 groups of splenocytes. Each group was then split into 3 equal aliquots and individually processed for (i) CD4+ T cell depletion or (ii) CD8+ T cell depletion or (iii) were not depleted. CD4+ or CD8+ T cells were depleted by positive selection using magnetic antibody cell separation (MACS) CD4 (L3T4) MicroBeads and CD8a (LY-2) MicroBeads with LD separation columns (Miltenyi) per the manufacturer’s instructions. The undepleted samples (not treated with MicroBeads) were run on MACS LD separation columns for control purposes. The column eluates were pelleted by centrifugation (450 × g) and then resuspended in RPMI-10. Mouse IFN-γ ELISpot assays were conducted as described above.
4
Single-cell RNA-seq of Foxp3+ T cells
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For RNAseq experiments, 8–10 week old Foxp3.RFP Rag.GFP mice were used. Single cell suspensions from thymi and spleens were prepared by smashing the organs through a plunger using 40 μm strainer. For thymi, samples were CD8 depleted using magnetic beads (CD8a Ly-2 Microbeads Miltenyi 130-117-044). For spleens, samples were CD4 enriched using magnetic beads (CD4 L3t4 Microbeads 130-117-043) following the manufacturer’s instructions. Alternatively, for RNAseq experiments from in vitro cultured cells (described in ‘In vitro IL18 culture’ section) cells were harvested from plates and washed in PBS twice.
The resulting single cells suspensions were then stained with fluorescently-labeled antibodies and DAPI (1:1000) 5 min, RT, before sorting by FACS Aria III cytometer. Cells were sorted directly unto lysis/binding buffer (Invitrogen 61012) and kept at −80°C until further processing of RNA.
The resulting single cells suspensions were then stained with fluorescently-labeled antibodies and DAPI (1:1000) 5 min, RT, before sorting by FACS Aria III cytometer. Cells were sorted directly unto lysis/binding buffer (Invitrogen 61012) and kept at −80°C until further processing of RNA.
5
Purification and Activation of CD8+ T Cells
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CD8+ T cells were purified from WT mice spleens by MACS with the CD8a+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). Purified cells were kept at rest for 12 hours in T cell media (RPMI with 10% FBS, 10 mM HEPES, 1% non-essential amino acids, 1 mM sodium pyruvate, 100 units/ mL penicillin, 100 units/ mL streptomycin and 55 nM β-ME). After 12 h, cells were stimulated with plate bound CD3ε (145-2C11, 2 μg/mL) and soluble αCD28 (37.51, 1 μg/mL) (both from BioXcell, West Lebanon, NH, USA) in T cell media supplemented with murine IL-2 (10 ng/mL, PeproTech, Rocky Hill, NJ, USA). Cells were collected at various time points. For in vivo Bcl3 kinetic analysis, CD8+ T cells were purified from LCMV infected or naïve mice spleen by CD8a (Ly-2) microbeads (Miltenyi Biotec, Auburn, CA, USA) at various time points. RNA was prepared by RNeasy mini kit and cDNAs were synthesized using QuantiTect reverse transcription kit (both from Qiagen, Hilden, Germany). Bcl3 levels were quantified relative to Gapdh by TaqMan assays on Quantstudio3 real time system (Applied Biosystems by Thermo Fisher Scientific, Carlsbad, CA, USA).
6
Generation of Primary Mouse Cytotoxic T Cells
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Spleen and lymph node cells were obtained from 8-to 14-week old OT-1 mice [23 (link)]. These mice have CD8+ T cells that recognize OVA257-264 in the context of H-2Kb. CD8+ cells were sorted using CD8a Ly-2 microbeads (Miltenyi 130-049-401) and stimulated in vitro with plate-bound anti-CD3/anti-CD28 or MEF.B7.OVA antigen presenting cells for 48–72hrs in complete media composed of RPMI 1640 medium with 10% FCS, sodium pyruvate, 50nM β-mercaptoethanol, penicillin, streptomycin and glutamine followed by expansion in rIL-2 (100 units/mL) for an additional 3–4 days to generate primary mouse CTLs. BI-141 murine hybridoma cells were maintained in complete media. OT-1 hybridoma cells were maintained as in [8 (link)]. MEF.B7.OVA cells stably expressing H-2Kb, B7.1, and OVA257-264 where maintained as in [5 (link)]. H-2Kb EG.7 cells constitutively expressing OVA257-264 and EL-4 cells were maintained in as in [24 (link)].
7
Isolation of CD8+ Splenocytes
8
Isolation and Activation of Murine CD8+ T Cells for CAR-T Cell Engineering
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CD8+ T lymphocytes were purified from spleens of C57/BL6 mice by positive selection with CD8a (Ly-2) Microbeads (Miltenyi Biotec) with high purity (90–95 %). T cells were activated with plate-bound anti-mouse CD3ε (2 µg/ml, clone 145-2C11) and anti-mouse CD28 (2 µg/ml clone 37.51) antibodies in presence of 50 U/ml IL-2. 24 h and 48 h after activation, T cells were transduced in retroviral supernatant supplemented with 10 µg/ml polybrene by centrifugation at 2500 rpm for 90 min at 32 °C. For expansion, CAR T cells were cultured in medium supplemented with 50 U/ml IL-2, IL-7 (10 ng/ml) and IL-15 (10 ng/ml). CAR-modified T cells were enriched by immunomagnetic selection using anti-mouse CD19-PE antibody (clone 1D3/CD19) and anti-PE microbeads (Miltenyi Biotec).
9
TH17 Cell Differentiation Protocol
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Spleen cells from healthy mice were depleted of CD8+ T lymphocytes using CD8a (Ly-2) MicroBeads (Miltenyi Biotec) and cultured in two sequential phases. For TGF-Beta-dependent TH17 differentiation, cells were cultured (5 × 106 cells/mL) with antibodies against CD3 (eBioscience; 10 µg/mL) and CD28 (eBioscience; 0.5 µg/mL) and the cytokines IL-6 (R & D Systems; 20 ng/mL) and TGF-β (R & D Systems; 1 ng/mL) in the presence or absence of IFN-β (100 U/mL) for three days. For IL-23-dependent TH17 differentiation, the cells initially cultured with IL-6 and TGF-β (without IFN-β) were washed and recultured for an additional three days with anti-CD3 (10 µg/mL), anti-CD28 (0.5 µg/mL), IL-23 (10 ng/mL) and anti-IFN-γ (10 µg/mL) with or without IFN-β (100 U/mL).
10
Purification and Fractionation of DP/CD8+ Cells
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Nuclear-cytosol fractionations of purified DP/CD8+ cells, with CD8a(Ly-2) microbeads (Miltenyi-Biotec) following manufacturer’s protocol, and of TALL-1 cells were performed as described [23 (link), 55 (link)].
WCE and immunoblotting assays were performed as described [23 (link)]. Used antibodies: Anti-Flag(F3165), anti-β-actin(A5441) (Sigma-Aldrich); anti-Phospho-β-arrestin1-Ser412(2416), anti-Notch3(2889) (Cell-Signaling); anti-β-arrestin-1(sc-9182), anti-Phospho-Erk(sc-7383), anti-Erk-1/2(sc-93/sc-154), anti-α-tubulin(sc-803) and anti-LaminB(sc6217) (Santa-Cruz); Anti-CXCR4 (ABCAM; 2074).
WCE and immunoblotting assays were performed as described [23 (link)]. Used antibodies: Anti-Flag(F3165), anti-β-actin(A5441) (Sigma-Aldrich); anti-Phospho-β-arrestin1-Ser412(2416), anti-Notch3(2889) (Cell-Signaling); anti-β-arrestin-1(sc-9182), anti-Phospho-Erk(sc-7383), anti-Erk-1/2(sc-93/sc-154), anti-α-tubulin(sc-803) and anti-LaminB(sc6217) (Santa-Cruz); Anti-CXCR4 (ABCAM; 2074).
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