CD8+ T cells were sorted from the spleens of WT or ICAM-1 KO C57BL/6 mice using mouse CD8 Microbeads and MACS LS columns (Miltenyi Biotec), according to the manufacturer's instructions. RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol (n = 3). RNA samples were processed for library preparation and sequenced using the BGISEQ-500 system at the Beijing Genomics Institute. Raw data were subjected to standard RNA sequencing analysis pipeline processing. HISAT2 and Bowtie2 were used to align the clean tag reads to the reference genome and reference genes. Matched reads were calculated and normalized using the RSEM software. Differential expression analysis of raw counts was conducted using the DESeq2 R library. Genes identified as significantly differentially expressed were characterized and analyzed at https://biosys.bgi.com . The adjusted P-value cut-offs for the lists of genes were as follows: ICAM-1 KO versus WT: 1402 differentially expressed genes with an adjusted P-value < 0.05 and fold change > 2. Volcano plots were created using the log2 fold change of genes determined as differentially expressed by DESeq2 (adjusted P-value < 0.05).
Mouse cd8 microbeads
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
Mouse CD8 Microbeads are a magnetic cell separation product designed for the isolation of CD8+ T cells from mouse cell samples. They consist of monoclonal antibodies specific for the CD8 surface marker conjugated to magnetic beads, allowing for the efficient separation and enrichment of CD8+ T cells from heterogeneous cell populations.
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Lab products found in correlation
Macs ls column, by Miltenyi Biotec (2 mentions)
Human cd8 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (1 mentions)
Clone 145 2c11, by BioLegend (1 mentions)
Clone 37, by BioLegend (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Cd25 microbeads, by Miltenyi Biotec (1 mentions)
7 protocols using mouse cd8 microbeads
1
CD8+ T Cell Transcriptome Analysis
2
Investigating Tumor Cell Lysis by Activated T Cells
3
Adoptive T Cell Therapy in Melanoma
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B16-F10 melanoma cells were injected subcutaneously (s.c.) into the backs of C57BL/6 mice (2 × 105 cells per mouse). Three days later, mice were injected intraperitoneally (i.p.) with 0, 150, or 300 mg/kg of CTX to induce lymphopenia. To prepare activated pmel-1 Thy1.1+CD8+ T cells, CD8+ T cells were isolated from the lymph nodes (LNs) and spleen of pmel-1 Thy1.1+ Tg mice using mouse CD8-microbeads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions, and activated in high glucose DMEM supplemented with 10% FBS and antibiotics in the presence of 5 μg of human gp100 (hgp100) peptide and 5% of CD8-depleted splenocytes for 2 days. Activated pmel-1 Thy1.1+CD8+ T cells were injected into mice via the intravenous (i.v.) route (2 × 106 cells per mouse) on day 5, and 25,000 IU of recombinant human IL-2 (rhIL-2) and/or 1 μg of IL7-Fc were administered daily to mice for 6 days.
4
Investigating Tumor Cell Lysis by Activated T Cells
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B16F10 tumor cells were cultured in complete DMEM medium as described above. Harvested B16F10 tumor cells were seeded in 24 or 96-well microplates or flasks in complete DMEM medium at 37°C for 2 days. Following aspiration of tumor culture medium, activated PMEL CD8+ T cells in resting phase as described above were suspended in complete RPMI medium supplemented with IL-2 (10 ng/mL) and added to the tumor cell culture at a T cell to tumor cell ratio of 1 to 1. After another 2-day co-culture, CD8+ T cells were isolated by Ficoll density gradient separation and MACS with mouse CD8 MicroBeads (Miltenyi Biotec) for Seahorse assay and flow cytometry analyses. To determine the lysis of targeT cells, the viability of tumor cells from the co-culture was measured with DAPI staining and flow cytometry. Similarly, human HER2 CAR-T cells were co-cultured with SKOV3-HER2 or ME275-HER2 tumor cells in vitro at an effector to target ratio of 1 to 1 in the presence or absence of IL-10/Fc (200 ng/mL) for 2 days. Human CD8+ HER2 CAR-T cells were isolated by Ficoll density gradient separation and MACS using the human CD8+ T cell isolation kit (Miltenyi Biotec) for Seahorse assay and flow cytometry analyses.
5
ICAM-1 Regulation of T-cell Proliferation
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CD8+ T cells were isolated from the spleens of ICAM-1 WT or ICAM-1 KO C57BL/6 mice using mouse CD8 Microbeads and MACS LS columns (Miltenyi Biotec) according to the manufacturer's instructions. Isolated T cells were suspended in PBS containing 5 μM CFSE (Thermo Fisher Scientific, Waltham, MA) and incubated at 37°C for 10 min. After washing, cells were resuspended in RPMI-1640 medium containing IL-2 (10 ng/mL). These CFSE-labeled T cells were then stimulated in plates precoated with anti-CD3 (1 µg/mL; clone 145-2C11, BioLegend) and anti-CD28 (2 µg/mL; clone 37.51, BioLegend) antibodies. Following a 3-day coculture, the cells were collected for flow cytometric analysis, and T-cell division was determined via CFSE dilution.
6
CD8+ T-Cell Cytotoxicity Assay
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Effector cells were isolated using mouse CD8 microbeads (Miltenyi Biotec) from peripheral blood. The CD8+ cells were incubated with cancer cell lines for 3.5 h at 37 °C. APC-conjugated CD107a mAb or isotype control rat IgG2a mAb were incubated in the mixture during the incubation period; after incubation, the cells were stained with additional PE-conjugated anti-CD8 mAb, FITC-conjugated anti-CD3εmAb, and 7-AAD Viability Dye and analyzed using FACSCanto II system and Flow-Jo software.
7
Isolation and Analysis of Murine CD8+ T Cells
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Nasal mucosa samples were processed into cell suspensions. Then the cell suspensions were centrifuged for 10 min at 200×g at 4 °C, and cells were harvested for flow cytometry analysis. CD8+ cells were isolated using mouse CD8 microbeads according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+CD25+ T cells were separated using an EasySep™ mouse CD8+ T cells enrichment kit (StemCell Technologies, Vancouver, BC, Canada), followed by isolation with CD25 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). For intracellular staining, these cells were stained with Foxp3 staining kit (MyBioSource, Inc., San Diego, CA, USA). Finally, above cells were resuspended and analyzed using a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJoSoftware (TreeStar Inc., Ashland, OR, USA).
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