Negative selection

Manufactured by STEMCELL

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Negative selection is a technique used to isolate specific cell populations from a heterogeneous mixture. It involves the removal of unwanted cells, allowing the desired cells to be obtained. The core function of negative selection is to selectively deplete target cells, resulting in the enrichment of the cell type of interest.

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55 protocols using negative selection

Activation and Isolation of T Cells

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All cells were grown at 37 °C under 5% CO₂. PBMCs were thawed in RPMI 1640 with 10% FBS, 1% penicillin/streptomycin, and rested overnight before stimulation with 15 ng/mL of anti-human CD3 antibody (clone OKT3, BioLegend) for 3 d in base media (RPMI 1640 with 10% FBS, 1% penicillin/streptomycin) containing 250 U IL-2 and 5 ng/mL IL-7 (hereafter termed IL-2/IL-7 media). OKT3 was removed after 3 d of activation and cells were maintained in IL-2/7 media at 1 × 10⁶ cells/mL for 7 to 10 d before isolation of CD8⁺ T cells by negative selection (StemCell) for coculture experiments. Separately, autologous PBMCs were also used to isolate CD4⁺ T cells by negative selection (StemCell). CD4⁺ T cells were activated using Human T-Activator CD3/CD28 Dynabeads (Thermo Fisher Scientific) in RPMI 1640 with Glutamax, 10% FBS, and 1% penicillin/streptomycin and 30 U IL-2.
T2 cells were cultured in RPMI 1640 + Glutamax with 10% FBS, 1% penicillin/streptomycin. RPMI 6666 cells were grown in the same media and used as an additional A2⁺ cell line for negative-selection stages of phage panning.

Sengupta S., Board N.L., Wu F., Moskovljevic M., Douglass J., Zhang J., Reinhold B.R., Duke-Cohan J., Yu J., Reed M.C., Tabdili Y., Azurmendi A., Fray E.J., Zhang H., Hsiue E.H., Jenike K., Ho Y.C., Gabelli S.B., Kinzler K.W., Vogelstein B., Zhou S., Siliciano J.D., Sadegh-Nasseri S., Reinherz E.L, & Siliciano R.F. (2022). TCR-mimic bispecific antibodies to target the HIV-1 reservoir. Proceedings of the National Academy of Sciences of the United States of America, 119(15), e2123406119.

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Naive T Cell Proliferation Assay

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CD45RA-positive (CD45RA⁺) naive T cells were isolated via negative selection (StemCell, Vancouver) according to the manufacturer’s protocol and cocultured with MDMs from each control and experimental group. Specifically, isolated naive T cells were labeled with CellTRace CFSE cell proliferation dye (Thermo Fisher) per the manufacturer’s instructions. Naive T cells were then activated with soluble anti-CD3 and anti-CD28 antibodies (BD Biosciences) at 1 μg/mL and then cocultured with donor-matched MDMs that were previously skewed toward M2a or left unskewed and each treated with Atripla, Triumeq, or vehicle control. After 72 h, cells were collected, and CFSE intensity was assessed by flow cytometry using a BD LSRFortessa flow cytometer.

Wallace J., Gonzalez H., Rajan R., Narasipura S.D., Virdi A.K., Olali A.Z., Naqib A., Arbieva Z., Maienschein-Cline M, & Al-Harthi L. (2022). Anti-HIV Drugs Cause Mitochondrial Dysfunction in Monocyte-Derived Macrophages. Antimicrobial Agents and Chemotherapy, 66(4), e01941-21.

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ILC2 Transfer and Ovalbumin Immunization

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ILC2 were isolated as recently described (21 (link)). In brief, donor mice were hydrodynamically injected with 4 µg each of IL-25 and IL-33 vectors to induce ILC2. Three days postinjection (dpi), ILC2 were sort-purified from spleen and mesenteric lymph nodes (MLNs) and in vitro expanded as indicated above. OTII transgenic CD4⁺ T cells were purified by negative selection (Stemcell Technologies) according to the manufacturer’s instructions and assessed at ≥95% purity. One day prior to cell transfer, recipient mice were intraperitoneally (i.p.) immunized with 20 µg Ovalbumin (Invivogen) in 200 µl Imject Alum (Thermo Scientific). 2 × 10⁶ CD4⁺ OTII T cells were co-transferred intravenously (i.v.) with 1 × 10⁶ WT or Btn2a2^–/–ILC2 into 8–14-week-old recipient hosts as indicated. Mice were sacrificed 3 days post adoptive cell transfer, and spleens were harvested for flow cytometric analyses.

Frech M., Omata Y., Schmalzl A., Wirtz S., Taher L., Schett G., Zaiss M.M, & Sarter K. (2022). Btn2a2 Regulates ILC2–T Cell Cross Talk in Type 2 Immune Responses. Frontiers in Immunology, 13, 757436.

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Purification and Activation of Mouse T Cells

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CD3⁺ cells were purified from 2–4 months old mouse spleens using MojoSort cell isolation kit (BioLegend). T cell activation was performed using low endotoxin, azide-free (LEAF) purified anti-mouse CD3 (Clone 145–2C11; 5 μg/mL) coated to the plate and LEAF Purified anti-mouse CD28 (clone 37.51; 2 μg/mL) in complete RPMI media for 24 or 48 h. Cytokine production was measured by LEGENDplex and confirmed by ELISA.
For T cell proliferation, CD4 T cells were isolated from spleen and LN of 10-week-old mice using negative selection (StemCell Technologies, Vancouver, Canada). CellTrace CFSE-labeled T cells were cocultured with anti-CD3/CD28-coated Dynabeads (Life Technologies, Carlsbad, CA) at 1:1 ratio according to the manufacturer’s protocol in a U bottom 96-well plate for 96 h in presence of rmIL-2 (30 units/mL).

Hernández-Ruiz M., Othy S., Herrera C., Nguyen H.T., Arrevillaga-Boni G., Catalan-Dibene J., Cahalan M.D, & Zlotnik A. (2019). Cxcl17−/− mice develop exacerbated disease in a T cell-dependent autoimmune model. Journal of leukocyte biology, 105(5), 1027-1039.

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Neutrophil-PBMC Co-culture Protocol

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Neutrophils were purified from whole blood by negative selection (StemCell), and co-cultured with granulocyte-depleted PBMCs at a ratio of 1:10. Purity of neutrophils and PBMCs was verified by staining as described below.

Agashe C., Chiang D., Grishin A., Masilamani M., Jones S.M., Wood R.A., Sicherer S.H., Burks A.W., Leung D.Y., Dawson P., Sampson H.A, & Berin M.C. (2017). Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells. Journal of immunological methods, 449, 23-27.

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Neutrophil Enrichment from Casein-Induced Inflammation

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Casein (9%; Sigma-Aldrich) was injected i.p. 16 and 3 h before peritoneal lavage to induce sterile inflammation. Cell were recovered from the inflamed peritoneal cavity after lavage with 10 ml cold sterile PBS. Negative selection (STEMCELL Technologies) beads were used to enrich for neutrophils according to the manufacturer’s guidelines and generated >97% neutrophils. Neutrophils were plated at 2 × 10⁶ cells/ml RPMI (Life Technologies) for functional analysis.

Ratitong B., Marshall M.E., Dragan M.A., Anunciado C.M., Abbondante S, & Pearlman E. (2022). Differential Roles for IL-1α and IL-1β in Pseudomonas aeruginosa Corneal Infection. Journal of immunology (Baltimore, Md. : 1950), 209(3), 548-558.

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Neutrophil Adhesion Assay: ICAM-1 and CXCL1

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Neutrophils obtained after 4-day differentiation (“in vitro-derived”) were washed and labeled using CFSE (BioLegend). Murine bone marrow neutrophils were isolated by negative selection (StemCell Technologies) and immediately labeled alongside in vitro-derived neutrophils using CFSE. 96-well plates were coated for 1 hour at room temperature or overnight at 4 °C with 2.5, 5, or 7.5 µg/mL ICAM-1 and/or 2.5 µg/mL CXCL1 in phosphate buffered saline (PBS) and then blocked with 1% casein (ThermoFisher) or 0.5% polyvinylpyrrolidone (Sigma Aldrich) in PBS for 2 hours at room temperature. Neutrophils were loaded into the 96-well plate at 0.5 × 10⁶ neutrophils per well in Hank’s balanced salt solution containing Ca²⁺ and Mg²⁺ (HBSS⁺⁺), and then incubated at 37 °C for 35 or 65 minutes. Following incubation, neutrophils were quantified by a plate reader for fluorescence intensity (CFSE), before and after sequential gentle washes with HBSS⁺⁺. The number of adherent neutrophils was inspected visually by light microscopy to corroborate with plate reader signal intensity. Each group was replicated in three to six wells per independent experiment.

Wilson Z.S., Witt H., Hazlett L., Harman M., Neumann B.M., Whitman A., Patel M., Ross R.S., Franck C., Reichner J.S, & Lefort C.T. (2020). Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking. Scientific Reports, 10, 2142.

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Isolation and Purification of Mouse Neutrophils

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Mouse bone marrow cells were isolated from femurs and tibiae. Polymorphonuclear cells (PMN) were purified by negative selection (Stemcell Technologies) according to the manufacturer's directions. For inflammatory neutrophils, mice were injected intraperitoneally (i.p.) with 1 ml of 4% of thioglycolate, 24 h later peritoneal contents were collected and neutrophils were purified by positive selection using biotinylated anti-Ly-6G (Biolegend) and MACS streptavidin-microbeads (Miltenyi Biotec), following the manufacturer's instructions.

Robledo-Avila F.H., Ruiz-Rosado J.D., Brockman K.L, & Partida-Sánchez S. (2020). The TRPM2 Ion Channel Regulates Inflammatory Functions of Neutrophils During Listeria monocytogenes Infection. Frontiers in Immunology, 11, 97.

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Quantifying HIV Reservoir in CD4+ T Cells

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The size of the HIV reservoir was estimated by measuring the frequency of CD4+ T cells harboring integrated HIV DNA, as previously described.^{30 (link)} Briefly, CD4+ T cells were isolated from cryopreserved PBMCs by negative selection (StemCell) and subjected to an Alu gnested-PCR to quantify the number of integrated HIV genomes.

Tawakol A., Ishai A., Li D., Takx R.A., Hur S., Kaiser Y., Pampaloni M., Rupert A., Hsu D., Sereti I., Fromentin R., Chomont N., Ganz P., Deeks S.G, & Hsue P.Y. (2017). Arterial and Lymph Node Inflammation Associate with Distinct Inflammatory Pathways in HIV. JAMA cardiology, 2(2), 163-171.

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Isolation and Purification of PBMC

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Peripheral blood mononuclear cells (PBMC) were isolated from blood by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden) and cryopreserved in 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) with 90% fetal bovine serum (FBS; Wisent, Inc. St. Bruno, QC, Canada). CD4 T cells were isolated from thawed PBMC by positive selection using immunomagnetic beads (STEMCELL Technologies, Inc. Vancouver, BC, Canada). The purity of the CD4 cell population was verified by flow cytometry (average 95.3%). NK cells were isolated from thawed PBMC by negative selection (STEMCELL Technologies, Inc.) and yielded an average purity of 97.2%.

Song R., Lisovsky I., Lebouché B., Routy J.P., Bruneau J, & Bernard N.F. (2014). HIV Protective KIR3DL1/S1-HLA-B Genotypes Influence NK Cell-Mediated Inhibition of HIV Replication in Autologous CD4 Targets. PLoS Pathogens, 10(1), e1003867.

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