Negative selection kit

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The Negative selection kit is a laboratory tool used to isolate specific cell types from a mixed cell population. It functions by removing unwanted cells, allowing the desired cell type to be collected. The kit contains the necessary reagents and components to perform this cell separation process efficiently.

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47 protocols using negative selection kit

Isolation and Characterization of NK Cells from Peripheral Blood

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Peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficoll-Hypaque (GE Healthcare). From the peripheral blood mononuclear cells, NK cells were isolated by a negative selection kit according to the manufacturer’s instructions (Miltenyi Biotec). Isolated NK cells were frozen in liquid nitrogen and stored for deoxyribonucleic acid (DNA) extraction at a later date. DNA from NK cells was extracted using the QIAamp DNA extraction kit (Qiagen) according to manufacturer’s instructions, and the concentration and quality of each DNA extraction was assessed using the NanoDrop Spectrophotometer 1000 (NanoDrop Technologies). Prior to genetic typing, NK cell DNA was stored at −20 °C.

Huth T.K., Brenu E.W., Staines D.R, & Marshall-Gradisnik S.M. (2016). Killer Cell Immunoglobulin-like Receptor Genotype and Haplotype Investigation of Natural Killer Cells from an Australian Population of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients. Gene Regulation and Systems Biology, 10, 43-49.

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SARS-CoV-2 Spike Antibody-Mediated NK Cell Response

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Human NK cells were enriched from fresh peripheral blood of healthy human volunteer donors’ buffy coats, obtained by Clínica Sanatorio Alemán blood bank at Concepción, Chile.
Briefly, a negative selection kit (Miltenyi) was used to isolate NK cells. 2 µg /ml of SARS-CoV-2 spike recombinant proteins were coated on ELISA High Bind Microplate (Corning) at 4°C overnight, and plates were blocked with 5% bovine serum albumin (BSA) prior to addition of the serum dilutions (1/100) in PBS for 2 hours at 37°C. Unbound antibodies were removed by washing wells 3X with PBS prior to the addition of NK cells. The NK cells were added at 5 x 10⁵ cells/well in the presence of brefeldin A (BioLegend), monensin (BioLegend), and anti-CD107a phycoerythrin (PE) (BioLegend) and incubated for 5 hours at 37°C. NK cells were surface stained with CD56 Alexa Fluor 647 (BioLegend), followed by intracellular staining with IFNγ Alexa Fluor 488 (BioLegend) and MIP1β Brilliant Violet 421 (BioLegend) using the Fix & Perm cell permeabilization kit (Invitrogen) used to detect the production of cytokine/chemokine. Cells were analyzed on a BD LSRFortessa X-20 flow cytometer and data was analyzed using FlowJo software (37 (link)).

Fuentes-Villalobos F., Garrido J.L., Medina M.A., Zambrano N., Ross N., Bravo F., Gaete-Argel A., Oyarzún-Arrau A., Amanat F., Soto-Rifo R., Valiente-Echeverría F., Ocampo R., Esveile C., Ferreira L., Cabrera J., Torres V., Rioseco M.L., Riquelme R., Barría S., Alvarez R., Pinos Y., Krammer F., Calvo M, & Barria M.I. (2022). Sustained Antibody-Dependent NK Cell Functions in Mild COVID-19 Outpatients During Convalescence. Frontiers in Immunology, 13, 796481.

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Naïve T-cell activation by treated DCs

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Naïve CD4 + T-cells were isolated from spleen using the negative selection kit (Miltenyi Biotec, The Netherlands). A total of 1 × 10⁵ purified naïve T-cells were co-cultured with treated DCs at a 1:10 ratio in a 96-well U-bottom culture plates for 6 days. T-cell subpopulations were then stained with antibodies and identified by flow cytometry, the gating strategy of T cells subpopulations is shown in Fig. S10. Supernatant was collected at day 6.

Xiao L., van’t Land B., Engen P.A., Naqib A., Green S.J., Nato A., Leusink-Muis T., Garssen J., Keshavarzian A., Stahl B, & Folkerts G. (2018). Human milk oligosaccharides protect against the development of autoimmune diabetes in NOD-mice. Scientific Reports, 8, 3829.

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Quantifying Latent HIV Reservoir

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The levels of latently infected CD4 T cells in blood and spleen were assessed by CD4 T cells QVOA as previously described (4 (link), 39 (link)). In brief, bulk CD4 T cells were isolated using the negative selection kit (Miltenyi) and plated in fivefold limiting dilutions. Cells were activated by being cocultured with CEMsx174 cells for 10 days. On day 10, supernatants were harvested and assessed for RNA, and the frequency of cells harboring replication-competent virus were determined as previously described (86 (link)).

Abreu C.M., Veenhuis R.T., Avalos C.R., Graham S., Parrilla D.R., Ferreira E.A., Queen S.E., Shirk E.N., Bullock B.T., Li M., Metcalf Pate K.A., Beck S.E., Mangus L.M., Mankowski J.L., Mac Gabhann F., O’Connor S.L., Gama L, & Clements J.E. (2019). Myeloid and CD4 T Cells Comprise the Latent Reservoir in Antiretroviral Therapy-Suppressed SIVmac251-Infected Macaques. mBio, 10(4), e01659-19.

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Immune Cell Isolation Protocol

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Tissues were resected and mechanically disaggregated. Spleens were further digested with collagenase D (Roche) and centrifuged on an M-lympholyte gradient (Cedarlane). Tumors were digested with collagenase IV, hyaluronidase, and deoxyribonuclease (Sigma) at 37°C for 3 hours. DCs were purified using CD11c microbeads and naïve CD4⁺ T cells isolated with a negative selection kit (Miltenyi Biotec).

Holtzhausen A., Zhao F., Evans K.S., Tsutsui M., Orabona C., Tyler D.S, & Hanks B.A. (2015). Melnoma-Derived Wnt5a Promotes Local Dendritic-Cell Expression of IDO and Immunotolerance: Opportunities for Pharmacologic Enhancement of Immunotherapy. Cancer immunology research, 3(9), 1082-1095.

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Generation of Monocyte-Derived Dendritic Cells

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Peripheral blood samples obtained from human subjects were free of Hp infection as determined by using the carbon 14 urea breath test (C14 UBT). CD14⁺ cells purified by sorting using anti-CD14-labeled magnetic beads (MACS, Miltenyi, Germany) from peripheral blood of healthy donors were cultured in the presence of GM-CSF (50 ng/ml; PeproTech, Rocky Hill, NJ, USA) and IL-4 (10 ng/ml; PeproTech, Rocky Hill, NJ, USA) to generate monocyte-derived DCs. Autologous CD4⁺ T cells were isolated from the PBMCs using a negative selection kit (MACS Miltenyi Biotec, GmbH, Germany) and activated with a combination of anti-CD3 (5 ug/ml; eBioscience, San Diego, CA, USA) and anti-CD28 Abs (2 ug/ml; eBioscience, San Diego, CA, USA).

Chang L.L., Hsu W.H., Kao M.C., Chou C.C., Lin C.C., Liu C.J., Weng B.C., Kuo F.C., Kuo C.H., Lin M.H., Wang C.J., Lin C.H., Wu D.C, & Huang S.K. (2018). Stromal C-type lectin receptor COLEC12 integrates H. pylori, PGE2-EP2/4 axis and innate immunity in gastric diseases. Scientific Reports, 8, 3821.

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Isolation of CD14+ Monocytes from PBMC

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Peripheral blood was obtained from venipuncture from four donors upon informed consent. Peripheral blood mononuclear cells (PBMC) were obtained as described [23 (link)]. Briefly, blood was layered above a high-density glucose solution, followed by centrifugation without a break, PBMC collection, and hypotonic lysis of contaminating red cells. Subsequently, CD14⁺ monocytes were isolated by magnetic bead purification based on a negative selection kit (Miltenyi Biotec, Teterow, Germany).

Bekeschus S., Ispirjan M., Freund E., Kinnen F., Moritz J., Saadati F., Eckroth J., Singer D., Stope M.B., Wende K., Ritter C.A., Schroeder H.W, & Marx S. (2022). Gas Plasma Exposure of Glioblastoma Is Cytotoxic and Immunomodulatory in Patient-Derived GBM Tissue. Cancers, 14(3), 813.

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Adoptive T cell transfer and influenza infection

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CD4⁺ T cells were isolated from WT CD45.1⁺ and Arg1 CKO (CD45.2⁺) mouse spleens using a negative selection kit from Stem Cell Technologies ( #19852) according to the manufacturer’s protocol. Naı¨ve CD8⁺ T cells from WT spleens were isolated using a negative selection kit from Miltenyi Biotech (#130–096-543). WT and Arg1 CKO CD4⁺ T cell were combined at a 50:50% ratio and then combined with WT naı¨ve CD8⁺ T cells and injected i.v. into Rag1 KO mice. 7 days after cell transfer the mice were infected i.n. with PR8–33. 7 days after infection the lungs were perfused and collected for enumeration of cell frequencies.

West E.E., Merle N.S., Kamiński M.M., Palacios G., Kumar D., Wang L., Bibby J.A., Overdahl K., Jarmusch A.K., Freeley S., Lee D.Y., Thompson J.W., Yu Z.X., Taylor N., Sitbon M., Green D.R., Bohrer A., Mayer-Barber K.D., Afzali B., Kazemian M., Scholl-Buergi S., Karall D., Huemer M, & Kemper C. (2023). Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection. Immunity, 56(9), 2036-2053.e12.

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Adoptive Transfer of CD4+ T Cells

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Ten to 12-week-old WT C57BL/6J mice were injected IV with 10⁵ FFU of ZIKV or PBS as a negative control. 30 DPI, splenocytes were harvested and prepared for use in a Milteyni enrichment kit. CD4⁺T cells were purified to 97% purity using a Miltenyi negative selection kit (139-104-454) according to manufacturer’s instructions. ~3x10⁶ cells were administered to Ifnar1^-/- mice IV route 1 day prior to infection.

Hassert M., Wolf K.J., Schwetye K.E., DiPaolo R.J., Brien J.D, & Pinto A.K. (2018). CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection. PLoS Pathogens, 14(9), e1007237.

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Tracking Adoptive Transfer of Naive CD4+ T Cells in BCG-Vaccinated Mice

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Naïve CD4⁺ T cells from P25 TCR-Tg/GFP mice were purified by negative selection with the Miltenyi Biotec negative selection kit as mentioned above, with slight modification by adding anti-CD44 conjugated to biotin (Clone IM7) (ThermoFisher Scientific, Waltham, MA, USA) to remove small numbers of memory T cells that are present in these animals. Purified CD4⁺ T cells (4 x 10⁴ in 100 μl of PBS) were injected intravenously into wild type C57BL/6 mice. Sixteen hours later, 5 x 10⁶ CFU of BCG were administered in a volume of 100 μl of PBS subcutaneously at the base of the tail. On day 7 after vaccination, mice were euthanized and spleens were harvested and analyzed by FACS. Tissues were also fixed in 10% neutral buffered formalin (NBF), paraffin embedded, and sectioned. Antigen retrieval with 10 mM Na citrate pH 6.0 was performed. Sections were blocked in 5% donkey serum / 2% BSA and stained with rabbit anti-GFP antibody (A11122; ThermoFisher Scientific, Waltham, MA, USA) followed by biotin-conjugated anti-rabbit IgG antibody. Avidin-peroxidase complex was added and visualized by the addition of the DAB (3,3’-Diaminobenzidine) chromogenic substrate. Sections were counterstained with hematoxylin and images were acquired with a Pannoramic 250 Flash II slide scanner (3DHistec, Ltd.).

Ng T.W., Wirchnianski A.S., Wec A.Z., Fels J.M., Johndrow C.T., Saunders K.O., Liao H.X., Chan J., Jacobs WR J.r., Chandran K, & Porcelli S.A. (2020). Exploiting pre-existing CD4+ T cell help from BCG vaccination to improve anti-viral antibody responses. Journal of immunology (Baltimore, Md. : 1950), 205(2), 425-437.

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