Cd69 microbead kit 2, by Miltenyi Biotec - Product details

1

Flow Cytometry Isolation of CD4+ T Cell Subsets

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PBMCs were isolated from leukapheresis samples by Ficoll density centrifugation. CD4⁺ cells were isolated from PBMCs using the EasySep™ Human CD4⁺ T-cell Isolation Kit (Stemcell Technologies). Resting CD4⁺ cells were then isolated by negative depletion of cells expressing CD69, CD25, or HLA-DR (CD69 MicroBead Kit II, CD25 MicroBeads, Anti-HLA-DR MicroBeads; Miltenyi Biotec).
Cells were stained with CD27-BV421, CD3-BV510, CCR7-PE, CD45RO-APC, CD4-PECy7 (Biolegend) and sorted on a MoFlo Legacy (Beckman Coulter). Viability was determined using a propidium iodide stain. Cells were sorted from gated live singlet CD3⁺CD4⁺ lymphocytes based on the following expression patterns: (TN)CD45RO⁻/CCR7⁺/CD27⁺, (TCM)CD45RO⁺/CCR7⁺/CD27⁺, (TTM)CD45RO⁺/CCR7⁻/CD27⁺, (TEM)CD45RO⁺/CCR7⁻/CD27⁻.

Kwon K.J., Timmons A.E., Sengupta S., Simonetti F.R., Zhang H., Hoh R., Deeks S.G., Siliciano J.D, & Siliciano R.F. (2020). Different human resting memory CD4+ T cell subsets show similar low inducibility of latent HIV-1 proviruses. Science translational medicine, 12(528), eaax6795.

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2

Isolation of Resting CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences; Marlborough, MA) per manufacturer’s instructions. Untouched total CD4⁺ T-cells were then enriched from PBMC using negative immunomagnetic selection via the EasySep Human CD4⁺ T-Cell Enrichment Kit (StemCell Technologies; Vancouver, BC). In some experiments, resting CD4⁺ T-cells (CD4⁺, CD69⁻, CD25⁻ and HLA-DR⁻) were isolated using a second negative selection step (CD25-Biotin; Anti-Biotin MicroBeads; CD69 MicroBead Kit II; Anti–HLA-DR MicroBeads, all from Miltenyi Biotec). Resting CD4⁺ T-cell purity was consistently >95% as assessed using flow cytometry.

Bruner K.M., Wang Z., Simonetti F.R., Bender A.M., Kwon K.J., Sengupta S., Fray E.J., Beg S.A., Antar A.A., Jenike K.M., Bertagnolli L.N., Capoferri A.A., Kufera J.T., Timmons A., Nobles C., Gregg J., Wada N., Ho Y.C., Zhang H., Margolick J.B., Blankson J.N., Deeks S.G., Bushman F.D., Siliciano J.D., Laird G.M, & Siliciano R.F. (2019). A novel quantitative approach for measuring the reservoir of latent HIV-1 proviruses. Nature, 566(7742), 120-125.

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3

Isolation of Resting CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences; Marlborough, MA) per manufacturer’s instructions. Untouched total CD4⁺ T-cells were then enriched from PBMC using negative immunomagnetic selection via the EasySep Human CD4⁺ T-Cell Enrichment Kit (StemCell Technologies; Vancouver, BC). In some experiments, resting CD4⁺ T-cells (CD4⁺, CD69⁻, CD25⁻ and HLA-DR⁻) were isolated using a second negative selection step (CD25-Biotin; Anti-Biotin MicroBeads; CD69 MicroBead Kit II; Anti–HLA-DR MicroBeads, all from Miltenyi Biotec). Resting CD4⁺ T-cell purity was consistently >95% as assessed using flow cytometry.

Bruner K.M., Wang Z., Simonetti F.R., Bender A.M., Kwon K.J., Sengupta S., Fray E.J., Beg S.A., Antar A.A., Jenike K.M., Bertagnolli L.N., Capoferri A.A., Kufera J.T., Timmons A., Nobles C., Gregg J., Wada N., Ho Y.C., Zhang H., Margolick J.B., Blankson J.N., Deeks S.G., Bushman F.D., Siliciano J.D., Laird G.M, & Siliciano R.F. (2019). A novel quantitative approach for measuring the reservoir of latent HIV-1 proviruses. Nature, 566(7742), 120-125.

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4

Isolation of Resting CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated using density centrifugation on a Ficoll-Hypaque gradient. CD4⁺ T cells were isolated from PBMCs using a negative selection method (CD4⁺ T cell Isolation Kit II, Miltenyi Biotec). Resting CD4⁺ lymphocytes (CD4⁺, CD69⁻, CD25⁻ and HLA-DR⁻) were enriched by a second negative depletion (CD25⁻Biotin; Anti-Biotin MicroBeads; CD69 MicroBead Kit II; Anti–HLA-DR MicroBeads, all from Miltenyi Biotec). Resting CD4⁺ cell purity was consistently >95% as assessed using flow cytometry.

Bruner K.M., Murray A.J., Pollack R.A., Soliman M.G., Laskey S.B., Capoferri A.A., Lai J., Strain M.C., Lada S.M., Hoh R., Ho Y.C., Richman D.D., Deeks S.G., Siliciano J.D, & Siliciano R.F. (2016). Defective proviruses rapidly accumulate during acute HIV-1 infection. Nature medicine, 22(9), 1043-1049.

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5

Isolation of Resting Memory CD4+ T Cells

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Total peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences). Total CD4⁺ T cells were then enriched from PBMCs using negative immunomagnetic selection with the EasySep Human CD4⁺ T Cell Enrichment Kit (StemCell Technologies). Resting memory CD4⁺ T cells (CD4⁺, CD69⁻, CD25⁻, HLA-DR⁻, and CD45RA⁻) were isolated using a second negative selection step (CD25-Biotin; Anti-Biotin MicroBeads; CD69 MicroBead Kit II; Anti–HLA-DR MicroBeads, Anti-CD45RA Microbeads; all from Miltenyi Biotec). Memory CD4⁺ T cell purity was >99% as assessed using flow cytometry specific for the CD4⁺, CD45RO⁺ population.

Kufera J.T., Armstrong C., Wu F., Singhal A., Zhang H., Lai J., Wilkins H.N., Simonetti F.R., Siliciano J.D, & Siliciano R.F. (2024). CD4+ T cells with latent HIV-1 have reduced proliferative responses to T cell receptor stimulation. The Journal of Experimental Medicine, 221(3), e20231511.

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6

Isolation of Resting CD4+ T Cells from PBMCs

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Bcl2 model cells were generated as previously described [53 (link)]. Peripheral blood mononuclear cells (PBMCs) from HIV negative and positive study participants were purified on Ficoll-paque PLUS (GE Healthcare, Cat#17-1440-02). CD4⁺ T lymphocytes were extracted (Miltenyi Biotec Cat#130-096-533) by negative selection. Resting CD4⁺ T cells were isolated by subsequent negative selection using CD25 MicroBeads II, CD69 MicroBead Kit II, and Anti-HLA-DR MicroBeads (Miltenyi Biotec Cat#130-092-983, Cat#130-092-355, Cat#130-046-101). Cells were kept in RPMI 1640 medium (Hyclone, Cat# SH30096_01), 10% FBS (Life Technologies, Cat# 10270–106), 1% Glutamax (Life Technologies, Cat# 35050), 1% Penicillin-streptomycin (Life Technologies, Cat# 15140–122). For active growth conditions, media was supplemented with human interleukin-2 IS (Miltenyi Biotec, Cat#130-097-742; Lot#5170516373) final concentration 100U/ml and 5% T-cell conditioned media, according to the protocol.

Lindqvist B., Svensson Akusjärvi S., Sönnerborg A., Dimitriou M, & Svensson J.P. (2020). Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells. PLoS Pathogens, 16(1), e1008264.

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Isolation of Resting CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated using density centrifugation on a Ficoll-Hypaque gradient. CD4⁺ T cells were isolated from PBMCs using a negative selection method (CD4⁺ T cell Isolation Kit II, Miltenyi Biotec). Resting CD4⁺ lymphocytes (CD4⁺, CD69⁻, CD25⁻ and HLA-DR⁻) were enriched by a second negative depletion (CD25⁻Biotin; Anti-Biotin MicroBeads; CD69 MicroBead Kit II; Anti–HLA-DR MicroBeads, all from Miltenyi Biotec). Resting CD4⁺ cell purity was consistently >95% as assessed using flow cytometry.

Bruner K.M., Murray A.J., Pollack R.A., Soliman M.G., Laskey S.B., Capoferri A.A., Lai J., Strain M.C., Lada S.M., Hoh R., Ho Y.C., Richman D.D., Deeks S.G., Siliciano J.D, & Siliciano R.F. (2016). Defective proviruses rapidly accumulate during acute HIV-1 infection. Nature medicine, 22(9), 1043-1049.

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8

Isolation of resting CD4+ T cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using FicollPaque PLUS (GE Healthcare Life Sciences) following manufacturer’s instructions. Total CD4⁺ T cells were then enriched from PBMCs using negative immunomagnetic selection using the EasySep Human CD4⁺ T-Cell Enrichment Kit (StemCell Technologies). Resting CD4+ T cells (CD69–, CD25– and HLA-DR–) were isolated using a second negative selection step (CD25-Biotin; Anti-Biotin MicroBeads; CD69 MicroBead Kit II; Anti–HLA-DR MicroBeads, all from Miltenyi Biotec).

White J.A., Kufera J.T., Bachmann N., Dai W., Simonetti F.R., Armstrong C., Lai J., Beg S., Siliciano J.D, & Siliciano R.F. (2022). Measuring the latent reservoir for HIV-1: Quantification bias in near full-length genome sequencing methods. PLoS Pathogens, 18(9), e1010845.

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9

Isolation of Resting CD4+ T Cells

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The Johns Hopkins Institutional Review Board approved this study and all research participants in this study gave written informed consent. Infected individuals were enrolled under the criteria of suppression of viremia to undetectable levels (<50 copies mL⁻¹) on ART for at least 6 months. PBMC were purified using density centrifugation from whole blood or continuous-flow centrifugation leukapheresis product. CD4⁺ T lymphocytes were enriched by negative depletion (CD4+ T cell Isolation Kit, Miltenyi Biotec). Resting CD4⁺ T lymphocytes were further enriched by depletion of cells expressing CD69, CD25, or HLA-DR (CD69 MicroBead Kit II, Miltenyi Biotec; CD25 MicroBeads, Miltenyi Biotec; Anti-HLA-DR MicroBeads, Miltenyi Biotec). Purity of resting CD4⁺ lymphocytes was verified by flow cytometry and was typically greater than 95%. With the exception of experiments designed to detect viral outgrowth, cells were cultured with 10 μM T20 to prevent new infection events.

Bullen C.K., Laird G.M., Durand C.M., Siliciano J.D, & Siliciano R.F. (2014). Novel ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nature medicine, 20(4), 425-429.

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Isolation of Resting CD4+ T Cells

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The Johns Hopkins Institutional Review Board approved this study and all research participants in this study gave written informed consent. Infected individuals were enrolled under the criteria of suppression of viremia to undetectable levels (<50 copies mL⁻¹) on ART for at least 6 months. PBMC were purified using density centrifugation from whole blood or continuous-flow centrifugation leukapheresis product. CD4⁺ T lymphocytes were enriched by negative depletion (CD4+ T cell Isolation Kit, Miltenyi Biotec). Resting CD4⁺ T lymphocytes were further enriched by depletion of cells expressing CD69, CD25, or HLA-DR (CD69 MicroBead Kit II, Miltenyi Biotec; CD25 MicroBeads, Miltenyi Biotec; Anti-HLA-DR MicroBeads, Miltenyi Biotec). Purity of resting CD4⁺ lymphocytes was verified by flow cytometry and was typically greater than 95%. With the exception of experiments designed to detect viral outgrowth, cells were cultured with 10 μM T20 to prevent new infection events.

Bullen C.K., Laird G.M., Durand C.M., Siliciano J.D, & Siliciano R.F. (2014). Novel ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nature medicine, 20(4), 425-429.

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Cd69 microbead kit 2

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13 protocols using cd69 microbead kit 2

Flow Cytometry Isolation of CD4+ T Cell Subsets

Isolation of Resting CD4+ T Cells

Isolation of Resting CD4+ T Cells

Isolation of Resting CD4+ T Cells

Isolation of Resting Memory CD4+ T Cells

Isolation of Resting CD4+ T Cells from PBMCs

Isolation of Resting CD4+ T Cells

Isolation of resting CD4+ T cells

Isolation of Resting CD4+ T Cells

Isolation of Resting CD4+ T Cells

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