Immunomagnetic negative selection

Manufactured by STEMCELL

Sourced in Canada

Immunomagnetic negative selection is a laboratory technique used to isolate specific cell types from a heterogeneous cell population. It involves the use of magnetic beads coated with antibodies that bind to unwanted cells, allowing the desired cells to be separated magnetically.

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Lab products found in correlation

Transwell insert, by Corning (1 mentions) Interleukin 2 (il 2), by Abcam (1 mentions) Chromium next gem single cell 3 reagent kits v3, by 10x Genomics (1 mentions) Chromium next gem chip g, by 10x Genomics (1 mentions) Pen strep, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Hmvec l, by Lonza (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Sabm medium, by Lonza (1 mentions) Gelatin, by American Type Culture Collection (1 mentions) Cd3 fitc, by BD (1 mentions) Cd4 buv395, by BD (1 mentions) Pd1 pecf594, by BD (1 mentions) 41bb percpef710, by Thermo Fisher Scientific (1 mentions) Live dead ghost dye 780, by Cytek Biosciences (1 mentions) Lag3 bv711, by BD (1 mentions) Tim3 apc, by Thermo Fisher Scientific (1 mentions) Ctla 4 pe, by Thermo Fisher Scientific (1 mentions) Cd8 buv805, by BD (1 mentions) Rainbow beads, by Spherotech (1 mentions)

Close spelling variants of this product

Negative selection (55 citations) Immunomagnetic negative selection kit (8 citations) Immunomagnetic selection (2 citations) EasySep immunomagnetic negative selection kits (2 citations)

6 protocols using immunomagnetic negative selection

Transwell Assay for Cancer Cell Migration

Cited 2 times

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The process of this experiment was implemented as described in former study.18 (link),19 (link) Briefly, T cells were isolated from PBMC through immunomagnetic negative selection (STEMCELL Technologies, Canada). Isolated T cells were pre-activated by 100ng/mL CD3 antibody (Abcam, Shanghai, China) and recombination 10ng/mL IL-2 (Abcam, Shanghai, China). Cancer cells were transfected and cultured for 24h. The supernatant of cancer cell medium was collected. Polycarbonate membranes and cancer cell conditioned medium was placed in the bottom of the well. T cells (5×10⁵) were seeded and cultured on Transwell inserts (diameter, 6.5 mm) containing 5- or 3-μm pore size (Costar, USA). After 2h of incubation, the Transwell inserts were lifted. The number of transitional cells was measured by a hemocytometer, cell migrated were normalized to NC group (cell migrated (%) = Cell migrated/Cell migrated in NC×100%). Each experiment was replicated triplicates.

Ye J., Chen X, & Lu W. (2021). Identification and Experimental Validation of Immune-Associate lncRNAs for Predicting Prognosis in Cervical Cancer. OncoTargets and therapy, 14, 4721-4734.

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Multimodal Profiling of T Cells

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T cells were isolated from HLAC using Immunomagnetic negative selection (StemCell Technologies), and dead cells were removed using a dead cells removal kit (Miltenyi Biotec). The cells were stained with a panel of TotalSeq-A human antibodies (BioLegend) according to manufacturer’s protocol. Stained cells were then loaded onto a Chromium Next GEM chip G and both ADT (Antibody Derived Tag) and GEX (Gene Expression) libraries were generated using Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (10X genomics) for next generation sequencing. A range of 11,000-13,000 events per sample were collected for the subsequent single-cell RNAseq analysis. The data was preprocessed in Cell Ranger for alignment against the human genome and further analyzed in SeqGeq ^®.

Luo X., Frouard J., Zhang G., Neidleman J., Xie G., Sheedy E., Roan N.R, & Greene W.C. (2022). Subsets of Tissue CD4 T Cells Display Different Susceptibilities to HIV Infection and Death: Analysis by CyTOF and Single Cell RNA-seq. Frontiers in Immunology, 13, 883420.

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Culturing Human Lung Cells and PBMCs

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Human alveolar epithelial cells (Cell Biologics, Accegen) were cultured using SABM medium (Lonza) supplemented with growth factor kit and 5% v/v fetal bovine serum (FBS) in a T-25 flask coated with Gelatin (ATCC) until they reach 90% confluency.
Human microvascular lung endothelial cells (HMVEC-L) (Lonza) were cultured in EBM-2 Basal Medium supplemented with EGM-2 MV Microvascular Endothelial Cell Growth Medium and 1% v/v Pen-Strep (ThermoFisher) according to manufacturer’s instructions.
Peripheral blood mononuclear cells (PBMC) were isolated from fresh human buffy coat (Research Blood Components) using immunomagnetic negative selection (Stem Cell Technologies) and cultured in RPMI-1640 (Gibco) supplemented with 10% v/v FBS (ThermoFisher) and 1% v/v Pen-Strep (ThermoFisher) or cryo-preserved in FBS containing 10% dimethyl sulfoxide (DMSO) before use.

Kerns S.J., Belgur C., Petropolis D., Kanellias M., Barrile R., Sam J., Weinzierl T., Fauti T., Freimoser-Grundschober A., Eckmann J., Hage C., Geiger M., Ng P.R., Tien-Street W., Manatakis D.V., Micallef V., Gerard R., Bscheider M., Breous-Nystrom E., Schneider A., Giusti A.M., Bertinetti-Lapatki C., Grant H.S., Roth A.B., Hamilton G.A., Singer T., Karalis K., Moisan A., Bruenker P., Klein C., Bacac M., Gjorevski N, & Cabon L. (2021). Human immunocompetent Organ-on-Chip platforms allow safety profiling of tumor-targeted T-cell bispecific antibodies. eLife, 10, e67106.

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Adoptive Transfer of OT-1 T Cells

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For adoptive transfer of OT-1 T cells into B6 mice, OT-1 splenocytes were harvested as described above. CD8 T cells were isolated using immunomagnetic negative selection (StemCell, Vancouver, Canada; 19853), rinsed and suspended in PBS, and 2 × 10⁶ cells were adoptively transferred into 6–10 wk old, female, B6 mice via intraperitoneal injection. The day following transfer, mice were immunized subcutaneously with an individual SIINFEKL APL (100 µg) in complete Freund’s adjuvant (Sigma, F5881) or vehicle. Mice were euthanized at the times indicated, spleens were collected, processed as described above, and analyzed via flow cytometry using the following antibodies: SIINFEKL H2K^b tetramer-BV421 (NIH Tetramer Core Facility), CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BUV805 (BD 564920), LAG3-BV711 (BD 563179), PD1-PECF594 (BD 562523), TIM3-APC (eBioscience 17-5871-82), 41BB-PerCPeF710 (eBioscience 46-1371-82), CTLA4-PE (eBioscience 12-1529-42), CD44-BV786 (BD 563736) and Live/Dead Ghost dye 780 (Tonbo 13-0865-T100) or corresponding fluorescently labeled IgG controls.
Data collected on different days was normalized using rainbow beads (Spherotech, Lake Forest, IL; RFP-30-5A).

Zahm C.D., Colluru V, & McNeel D.G. (2017). Vaccination with High-Affinity Epitopes Impairs Antitumor Efficacy by Increasing PD-1 Expression on CD8+ T Cells. Cancer immunology research, 5(8), 630-641.

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Mouse NK Cell Isolation and Culture

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Mouse NK cells were isolated by negative immunomagnetic selection (Stem cell Technologies). The cells were cultured in a 5% CO₂ incubator at 37°C in culture media containing 5000 IU/ml IL-2 for 10 d. The culture medium used was RPMI-1640 supplemented with 10% FBS, 0.25 μg/ml amphotericin B, 10 U/ml penicillin G, 100 μg/ml streptomycin, 1 mM L-glutamine, 1% nonessential amino acids (Invitrogen), and 50 μM 2-mercaptoethanol. Purity of NK cells was determined by FACS analysis. The murine melanoma B16F10 and lymphoma EL4 and YAC-1 cells were purchased from ATCC (Manassas, VA).

Rah S.Y., Kwak J.Y., Chung Y.J, & Kim U.H. (2015). ADP-ribose/TRPM2-mediated Ca2+ signaling is essential for cytolytic degranulation and antitumor activity of natural killer cells. Scientific Reports, 5, 9482.

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Isolation and FACS Sorting of Tregs and Teffs

Cited 1 time

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Tregs and CD4⁺ Teffs were freshly isolated from buffy coats obtained from 13 volunteer blood donors according to our previously described protocol. Briefly, on the day of blood donation peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by Ficoll/Uropoline gradient centrifugation and subjected to negative immunomagnetic selection (StemCell Technologies, Canada). Subsequently, CD4⁺ T cells were stained with monoclonal antibodies (mAb) specific for the following antigens: CD3, CD4, CD25, CD127, CD8, CD19, CD16 and CD14. The last 4 mAbs were conjugated with the same fluorochrome in aim to cut-off in one step cytotoxic T cells (Tc), B cells, natural killer (NK) cells and monocytes, respectively, that could potentially contaminate isolated CD4⁺ population. These cells were defined all together in sorting algorithm as lineage. Then, cells were sorted with FACS sorter Influx (BD Biosciences, USA) into the following phenotype of Tregs: CD3⁺CD4⁺CD25^HighCD127^−/Lowlin⁻doublet^- and Teffs: CD3⁺CD4⁺CD25^-CD127^Highlin⁻doublet^- as it was reported before^{4 (link)–7 (link),14 (link),60 (link)}.

Marek-Trzonkowska N., Piekarska K., Filipowicz N., Piotrowski A., Gucwa M., Vogt K., Sawitzki B., Siebert J, & Trzonkowski P. (2017). Mild hypothermia provides Treg stability. Scientific Reports, 7, 11915.

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