Non human primate cd4 t cell isolation kit

Manufactured by Miltenyi Biotec

The Non-human primate CD4+ T cell isolation kit is a laboratory tool designed for the isolation of CD4+ T cells from non-human primate samples. The kit utilizes magnetic beads coated with antibodies specific to the CD4 surface marker to selectively capture and separate CD4+ T cells from the sample.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Anti human cd3, by BD (2 mentions) Anti human cd28, by BD (2 mentions) Dneasy extraction kit, by Qiagen (1 mentions) High pure viral rna kit, by Roche (1 mentions) Human il 15, by Miltenyi Biotec (1 mentions) Automated dna sequencer, by Thermo Fisher Scientific (1 mentions) Live dead aqua, by BD (1 mentions) Cd4 bv650, by BioLegend (1 mentions) Facsaria lsr 2, by BD (1 mentions) Ccr7 pe cy7 clone 3d12, by BD (1 mentions) Cd62l pe clone sk11, by BD (1 mentions) Deae dextran, by Merck Group (1 mentions) Rpmi medium, by Thermo Fisher Scientific (1 mentions) Recombinant human il 2 protein, by R&D Systems (1 mentions) T cell activation expansion kit, by Miltenyi Biotec (1 mentions) Camp direct immunoassay kit, by Abcam (1 mentions)

7 protocols using non human primate cd4 t cell isolation kit

Proviral Sequencing of Primary CD4+ T Cells

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Primary CD4⁺ T cells were prepared by negative selection from macaque PBMCs using a non-human primate CD4⁺ T cell isolation kit (Miltenyi). Total cellular DNA was extracted from CD4⁺ T cells using DNeasy extraction kit (QIAGEN). The DNA corresponding to the number of CD4⁺ T cells indicated in S1 Table was subjected to nested PCR amplification of proviral gag, vif, and nef cDNA fragments (nucleotide numbers [nt] 1231–2958 for gag, nt 4829–7000 for vif, and nt 8677–10196 for nef in SIVmac239 [accession number M33263]) for direct sequencing using dye terminator chemistry and an automated DNA sequencer (Applied Biosystems) as previously described [25 (link)]. For macaques R06-037, R05-005, R07-001, and R07-006 at 2 years, total cellular DNAs were extracted not directly from CD4⁺ T cells but after 8 days of culture described below. Dominant non-synonymous mutations were determined. For virus recovery, 0.5–2 x 10⁶ CD4⁺ T cells were cultured in the presence of 10 ng/ml human interleukin-7 (IL-7) (Miltenyi) and 10 ng/ml human IL-15 (Miltenyi) for 8 days. Then, viral RNA was extracted from supernatants of CD4⁺ T-cell culture using the High Pure Viral RNA kit (Roche Diagnostics) and subjected to reverse transcription and nested PCR (RT-PCR) amplification of viral gag cDNA fragments.

Nomura T., Yamamoto H., Ishii H., Akari H., Naruse T.K., Kimura A, & Matano T. (2015). Broadening of Virus-Specific CD8+ T-Cell Responses Is Indicative of Residual Viral Replication in Aviremic SIV Controllers. PLoS Pathogens, 11(11), e1005247.

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Multiparameter Flow Cytometry for T Cell Analysis

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For cell sorting, peripheral CD4⁺ T cells were first enriched by negative selection with the use of magnetic beads and column purification (nonhuman primate CD4⁺ T cell isolation kit; Miltenyi). Enriched CD4⁺ T cells were then stained with viability dye (Live/Dead Aqua) and previously determined volumes of the following fluorescently conjugated MAbs: CD3-AF700 (clone SP34-2), CD8-APC-Cy7 (clone SK1), CD95-PE-Cy5 (clone DX2), CD62L-PE (clone SK11), and CCR7-PE-Cy7 (clone 3D12) from BD Biosciences; CD4-BV650 from BioLegend. Sorted live CD3⁺CD8-CD4⁺ populations were defined as follows: naive cells, CD62L⁺ CCR7⁺ CD95-; and memory, CD95⁺. Sorting was performed on a FACSAria LSR II (BD Biosciences) equipped with FACSDiva software.

Bricker K.M., Obregon-Perko V., Williams B., Oliver D., Uddin F., Neja M., Hopkins L., Dashti A., Jean S., Wood J.S., Ehnert S., Liang S., Vanderford T., Tharp G.K., Bosinger S.E., Schauer A.P., Mavigner M., Cottrell M.L., Margolis D., Dunham R.M, & Chahroudi A. (2022). Altered Response Pattern following AZD5582 Treatment of SIV-Infected, ART-Suppressed Rhesus Macaque Infants. Journal of Virology, 96(7), e01699-21.

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Expansion of SIV in Rhesus CD4+ T Cells

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PBMC was isolated from blood of rhesus macaques using Ficoll-Paque PLUS (GE Heathcare). For virus expansion, we followed the protocol developed by George Shaw (Li et al., 2016 (link)). Briefly, CD4 T cells isolated from human or rhesus PBMC using non-human primate CD4+ T Cell Isolation Kit (Miltenyi Biotec) were stimulated with T Cell Activation/Expansion Kit (Miltenyi Biotec). Four days after stimulation, cells were infected with viruses in the presence of 30 μg/ml of DEAE-Dextran (Sigma) for 4 hours with gentle mixing every 30 min. SIVsmE660 (2008) were expanded once on human CD4 T cells and SIVsmE660 (2015) were expanded once on rhesus CD4 T cells (matching the original innocula). After infection, cells were washed with medium three times and resuspended in RPMI medium (Thermo Fisher Scientific) supplemented with 15% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific) and Recombinant Human IL-2 Protein (R&D SYSTEMS) at 30 IU/ml. Viruses were harvested every three days and p27 concentration was measured by p27 Antigen Capture Assay (ABL, Inc).

Iwamoto N., Mason R., Hu J., Ransier A., Welles H., Song K., Douek D, & Roederer M. (2018). A high throughput lentivirus sieving assay identifies neutralization resistant Envelope sequences and predicts in vivo sieving. Journal of immunological methods, 464, 64-73.

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Naïve CD4+ T Cell Activation and Differentiation

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CD4⁺ T cells from rhesus macaque PBMCs were enriched using the Non-Human Primate CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). Naïve CD4⁺ T cells were isolated by flow cytometry as CD4⁺CD45RA⁺CD95⁻CD28⁺CCR7⁺ cells. Naïve CD4⁺ T cells (7.5 × 10⁴ cells/well) were activated by plate-bound anti-human-CD3 and anti-human-CD28 (both at 5 µg/ml, BD) and cultured with recombinant human/mouse/rat activin A (50 ng/ml) and/or recombinant human IL-12 (5 ng/ml) for 5 d. Phenotype was assessed by flow cytometry, following the staining panel in Supplementary Table 3.

Locci M., Wu J., Arumemi F., Mikulski Z., Dahlberg C., Miller A.T, & Crotty S. (2016). Activin A programs human TFH cell differentiation. Nature immunology, 17(8), 976-984.

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Naïve CD4+ T Cell Activation and Differentiation

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Locci M., Wu J., Arumemi F., Mikulski Z., Dahlberg C., Miller A.T, & Crotty S. (2016). Activin A programs human TFH cell differentiation. Nature immunology, 17(8), 976-984.

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Measuring PKA in CD4+ T Cells

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CD4+T cells were isolated from PBMCs using non-human primate CD4+ T Cell Isolation Kit (Miltenyi, German). PKA activity, concentration and cAMP concentration in untreated or treated PBMCs were detected using PKA kinase activity kit (ImmuneChem, Canada), PKA ELISA kit (mlBio, China) and cAMP direct immunoassay kit (Biovision, American) respectively. PKA activity was also measured in CD4+ T cells. All procedures were according to the manufacturer's instructions.

Tian R.R., Liu B.B., Zhao M.L., Cai Y.J, & Zheng Y.T. (2024). Increased cAMP-PKA signaling pathway activation is involved in up-regulation of CTLA-4 expression in CD4+ T cells in acute SIVmac239-infected Chinese rhesus macaques. Virus Research, 341, 199313.

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Isolation of CD4+ T Cells from Primate

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CD4⁺ T cells were isolated from blood or lymph nodes (LNs). PBMCs were isolated from blood using Ficoll-Hypaque density gradient centrifugation. LNs were cut in small pieces, passed through a 70 μm cell strainer and rinsed with cold PBS. CD4⁺ T cells were isolated (from fresh PBMC and mesenteric LNs (MLNs) for SHIV_AD8OE, SIVΔNef and SIVmac251 infected animals or frozen PBMC/LNMC for SIVmac239 and SIVmac239Nef_stop infected animals) by negative selection using the non-human primate CD4⁺ T cell isolation kit (Miltenyi). Freshly enriched CD4⁺ T cells were resuspended at 2 × 10⁶ cells/ml in complete cRPMI (complete RPMI, 10% fetal bovine serum, 100 U/ml Penicillium and 100 μg/ml Streptomycin, 10 mM Hepes, 2 mM L-glutamine). A small fraction of cells was used to assess the purity of enriched CD4⁺ T cells (generally greater than 96%) and the level of T cell activation by flow cytometry.

Frank I., Acharya A., Routhu N.K., Aravantinou M., Harper J.L., Maldonado S., Sole Cigoli M., Semova S., Mazel S., Paiardini M., Derby N., Byrareddy S.N, & Martinelli E. (2019). A Tat/Rev Induced Limiting Dilution Assay to Measure Viral Reservoirs in Non-Human Primate Models of HIV Infection. Scientific Reports, 9, 12078.

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