Negative selection kit

Manufactured by STEMCELL

Sourced in Canada

The Negative Selection Kit is a laboratory equipment product designed for the isolation of specific cell types from a heterogeneous cell population. The kit utilizes antibody-coated magnetic beads to deplete unwanted cells, allowing for the enrichment of the desired cell type.

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Lab products found in correlation

58 protocols using negative selection kit

Isolation and Co-culture of Immune Cells

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Frozen HMNCs were recovered and rested in complete RPMI supplemented with 2ng/mL IL-15 (Miltenyl Biotec) and 25ug/mL DNAse-I (Sigma-Aldrich) for 45 mins at 37°C in 5% CO₂. CD56⁺ NK cells were magnetically isolated from HMNCs using a negative selection kit (Stemcell, Vancouver, Canada) and from peripheral blood samples using a RosetteSep™ kit (Stemcell). Whole PBMCs were isolated by density centrifugation using Ficoll-Paque™ PLUS and CD3⁺ T cells were magnetically isolated from PBMCs using a negative selection kit (Stemcell). PBMCs or CD3⁺ T Cells were stimulated with anti-CD3 (5µg/mL; Tonbo Biosciences, California, USA) and anti-CD28 (2µg/mL; Tonbo Biosciences) and co-cultured with either hepatic or peripheral blood NK cells at a 1:1 (PBMC : NK) ratio in cRPMI supplemented with 2ng/mL IL-15, unless otherwise indicated. A monoclonal antibody (mAb) against CD160 (MBL, Massachusetts, USA) or an IgG isotype control (Biolegend, California, USA) was added at 10µg/mL to assess its role in NK cell killing. After 24 hr the percentage of dead T cells was assessed using the coculture flow cytometry panel (Supplementary Table 2).

Jameson G., Harmon C., Santiago R.M., Houlihan D.D., Gallagher T.K., Lynch L., Robinson M.W, & O’Farrelly C. (2022). Human Hepatic CD56bright NK Cells Display a Tissue-Resident Transcriptional Profile and Enhanced Ability to Kill Allogenic CD8+ T Cells. Frontiers in Immunology, 13, 921212.

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Splenic T Cell Isolation from Mice

Cited 2 times

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Splenic T cells were purified from adult (2-6 months) mice as previously described (3 (link), 9 (link)). Briefly, pan-T cells were isolated with a negative selection kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s protocol. Isolated cells were washed with serum-free RPMI 1640 (Life Technologies, Grand Island, NY) prior to use.

Arkee T., Hostager B.S., Houtman J.C, & Bishop G.A. (2021). TRAF3 in T cells restrains negative regulators of LAT to promote TCR/CD28 signaling. Journal of immunology (Baltimore, Md. : 1950), 207(1), 322-332.

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Splenic B cell isolation and FISH analysis

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Splenic B cells were isolated from naïve mice, purified by negative selection kit (Stem Cell Technologies, Canada), activated with anti-CD40 and IL4 as described previously [82 (link)], and collected 4 days after culture for metaphase preparation and FISH analysis. FISH analysis was performed with specific BAC probes as previously described [82 (link)] (see details in Additional file 1). Genomic DNA was isolated from tumor masses or normal tissues from control mice, and Southern blotting was performed as previously described [41 (link)].

Chen Z., Elos M.T., Viboolsittiseri S.S., Gowan K., Leach S.M., Rice M., Eder M.D., Jones K, & Wang J.H. (2016). Combined deletion of Xrcc4 and Trp53 in mouse germinal center B cells leads to novel B cell lymphomas with clonal heterogeneity. Journal of Hematology & Oncology, 9, 2.

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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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Isolation of Human Mononuclear Cells

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Studies were approved by the University of Illinois at Chicago (UIC) Institutional Ethics Review Board and all donors gave informed written consent. Human mononuclear cells were isolated by Histopaque gradient centrifugation and monocytes were isolated from human peripheral blood (PB) using a negative selection kit according to the manufacturer’s instruction (StemCell Technology) [36 (link)–43 (link)].

Umar S., Palasiewicz K., Meyer A., Kumar P., Prabhakar B.S., Volin M.V., Rahat R., Al-Awqati M., Chang H.J., Zomorrodi R.K., Rehman J, & Shahrara S. (2022). Inhibition of IRAK4 dysregulates SARS-CoV-2 spike protein-induced macrophage inflammatory and glycolytic reprogramming. Cellular and Molecular Life Sciences, 79(6), 301.

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Profiling CD4+ T cells in Healthy Men

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Male participants without serious cardiovascular diseases who underwent routine health examinations were recruited in the Outpatient Department of Ruijin Hospital in Shanghai Jiao Tong University School of Medicine. Peripheral blood samples were collected, and sera were used for ELISA. Peripheral CD4⁺ T cells were sorted using a negative selection kit (17952, Stem cell, Vancouver, Canada) for gene expression, prostaglandin, and Western analyses. The study was reviewed and approved by the Human Ethics Committee of Shanghai Ruijin Hospital, and all participants provided written informed consent.

Kong D., Wan Q., Li J., Zuo S., Liu G., Liu Q., Wang C., Bai P., Duan S.Z., Zhou B., Gounari F., Lyu A., Lazarus M., Breyer R.M, & Yu Y. (2020). DP1 Activation Reverses Age-Related Hypertension Via NEDD4L-Mediated T-Bet Degradation in T Cells. Circulation, 141(8), 655-666.

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Isolation and Activation of Naive CD8+ T Cells

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Buffy coats of healthy donors were purchased from Gulf Coast Regional Blood Center. Informed consent was obtained for all subjects. PBMCs were isolated from blood by density gradient cell separation. Naive CD8⁺ T cells were isolated from PBMCs using a negative selection kit (STEMCELL) and stimulated with human T-activator CD3/CD28 Dynabeads (Invitrogen) plus human IL-2 (10 ng/ml) with or without indicated treatments.

Ma X., Xiao L., Liu L., Ye L., Su P., Bi E., Wang Q., Yang M., Qian J, & Yi Q. (2021). CD36-mediated ferroptosis dampens intratumoral CD8+ T-cell effector function and impairs their antitumor ability. Cell metabolism, 33(5), 1001-1012.e5.

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Genetic Modification of CD8+ T Cells

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Human peripheral blood mononuclear cells (PBMCs) from two individual donors were purchased from AllCells. CD8⁺ T cells were enriched from these samples using a negative selection kit (STEMCELL Technologies). Isolated T cells were activated with αCD3/CD28 ImmunoCult reagent and grown in ImmunoCult-XF T cell Expansion Medium (STEMCELL Technologies) with the addition of 10 ng/ml IL-15 and 100 U/ml IL-2. To delete LAYN at the genomic level, we used a gRNA targeting exon 4 (single gRNA target sequence 5′-GGTCATGTACCATCAGCCAT-3′) and a nontargeting “scramble” control sequence (5′-GGTTCTTGACTACCGTAAT-3′); gRNAs were purchased from Integrated DNA Technologies. Recombinant Cas9 protein (UC Berkeley QB3 Macrolab) was combined with gRNA and introduced into primary T cells via electroporation as previously described (Schumann et al., 2015 (link); Roth et al., 2018 (link)). Cells were subsequently cultured for 4 d before analyzing or incorporating into functional assays.

Mahuron K.M., Moreau J.M., Glasgow J.E., Boda D.P., Pauli M.L., Gouirand V., Panjabi L., Grewal R., Luber J.M., Mathur A.N., Feldman R.M., Shifrut E., Mehta P., Lowe M.M., Alvarado M.D., Marson A., Singer M., Wells J., Jupp R., Daud A.I, & Rosenblum M.D. (2020). Layilin augments integrin activation to promote antitumor immunity. The Journal of Experimental Medicine, 217(9), e20192080.

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Isolation and Activation of Splenic B Cells

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Splenic B cells were isolated from wt naïve mice in either pure B6 or non-B6 genetic background that is the same as G1XP lymphomas. B cells were purified by negative selection kit (Stem Cell Technologies, Canada), activated with anti-CD40 and IL-4 as described previously [27 (link)], and collected 4 days after culture for genomic DNA isolation which were subject to NGS. In vivo immunization and GC B cell isolation were performed as described previously [27 (link)]. The GC B cells were isolated from non-B6 genetic background that is the same as G1XP lymphomas.

Chen Z., Gowan K., Leach S.M., Viboolsittiseri S.S., Mishra A.K., Kadoishi T., Diener K., Gao B., Jones K, & Wang J.H. (2016). Unexpected effects of different genetic backgrounds on identification of genomic rearrangements via whole-genome next generation sequencing. BMC Genomics, 17, 823.

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Neutrophil Isolation and Co-culture Assay

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Neutrophils were isolated from healthy donor blood (Research Blood Components, LLC) using a negative selection kit (StemCell Technologies, Inc.) according to the instructions from the manufacturer. Cells were stained with Hoechst 33342 (Thermo Fisher), washed, and resuspended at 40 × 10⁶ cells per ml for loading into the device. Devices were loaded with mono‐ or co‐cultures of A. graevenitzii and S. aureus as described above. Following thorough washing of the main channel, neutrophils were then loaded and imaging commenced.

Jalali F., Ellett F., Balani P., Duncan M.J., Dewhirst F.E., Borisy G.G, & Irimia D. (2021). No man's land: Species‐specific formation of exclusion zones bordering Actinomyces graevenitzii microcolonies in nanoliter cultures. MicrobiologyOpen, 10(1), e1137.

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