Zombienir

Manufactured by BD

The ZombieNIR is a near-infrared spectroscopy (NIRS) instrument designed for laboratory applications. It measures the absorption of near-infrared light by samples, providing information about their chemical composition and physical properties. The core function of the ZombieNIR is to perform non-invasive, real-time analysis of various materials and substances.

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Lab products found in correlation

Zombie nir, by BioLegend (3 mentions) Pan b cell isolation kit, by Miltenyi Biotec (1 mentions) Facsaria 3, by BD (1 mentions) Anti cd20 pecy7, by BD (1 mentions) Anti cd3 apc efluro 780, by Thermo Fisher Scientific (1 mentions) Anti cd8 apc efluro 780, by Thermo Fisher Scientific (1 mentions) Anti cd16 apc efluro 780, by Thermo Fisher Scientific (1 mentions) Anti cd14 apc efluro 780, by Thermo Fisher Scientific (1 mentions) Rnasin ribonuclease inhibitor, by Promega (1 mentions) Collagenase type 1, by Thermo Fisher Scientific (1 mentions) Anti mouse cd16 32, by BD (1 mentions) Calcein blue am, by Thermo Fisher Scientific (1 mentions) Papain based brain tumor dissociation kit, by Miltenyi Biotec (1 mentions) Tcl buffer, by Qiagen (1 mentions) Wash buffer, by BD (1 mentions) Cell activation cocktail, by BioLegend (1 mentions) Lsrfortessa, by BD (1 mentions) Golgistop, by BD (1 mentions) Cytofix cytoperm kit, by BD (1 mentions) Cd138 bv421, by BD (1 mentions)

4 protocols using zombienir

Isolation and Characterization of SARS-CoV-2 RBD-Specific B Cells

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As previously described (Robbiani et al., 2020 (link)), PBMCs were enriched for B cells via negative selection using a pan–B cell isolation kit (130-101-638; Miltenyi Biotec) according to the manufacturer’s protocol. Enriched B cells were incubated with fluorophore-labeled RBD and ovalbumin, and in the presence of antihuman antibodies anti-CD3-APC-eFluro 780 (47-0037-41; Invitrogen), anti-CD8-APC-eFluro 780 (47-0086-42; Invitrogen), anti-CD14-APC-eFluro 780 (47-0149-42; Invitrogen), anti-CD16-APC-eFluro 780 (47-0168-41; Invitrogen), anti-CD20-PECy7 (335793; BD Biosciences), and Zombie NIR (423105; BioLegend) in FACS buffer (1 × PBS, 2% calf serum, 1mM EDTA) for 30 min on ice. Single CD3⁻CD8⁻CD14⁻CD16⁻ZombieNIR^-CD20⁺Ova⁻RBD⁺RBD KEN⁺ were sorted using a FACS Aria III (Becton Dickinson) into individual wells of a 96-well plate, each containing 4 μl of lysis buffer comprising 0.5× PBS, 10 mM dithiothreitol, and 3,000 U/ml RNasin Ribonuclease Inhibitors (N2615; Promega). Sorted cells were frozen on dry ice and stored at −80°C until further processing.

Agudelo M., Muecksch F., Schaefer-Babajew D., Cho A., DaSilva J., Bednarski E., Ramos V., Oliveira T.Y., Cipolla M., Gazumyan A., Zong S., Rodrigues D.A., Lira G.S., Conde L., Aguiar R.S., Ferreira OC J.r., Tanuri A., Affonso K.C., Galliez R.M., Castineiras T.M., Echevarria-Lima J., Bozza M.T., Vale A.M., Bieniasz P.D., Hatziioannou T, & Nussenzweig M.C. (2022). Plasma and memory antibody responses to Gamma SARS-CoV-2 provide limited cross-protection to other variants. The Journal of Experimental Medicine, 219(9), e20220367.

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Single-Cell RNA-Seq of Brain Immune Cell Isolation

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All mice were perfused with ice-cold PBS after euthanasia. The collected brains were mechanically and enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec) supplemented with 0.1% typeI collagenase (Thermo Fisher Scientific) / PBS. Cells were first stained with calcein Blue AM (Life Technologies) and Zombie NIR (BioLegend) for 30min at 4 °C, and with anti-mouse CD16/32 (BD Biosciences) for 5min. After washing cells with ice-cold 2% FBS / PBS, cells were then stained with anti-mouse CD45-PerCP (clone 30-F11, BD Biosciences) for 30min at 4 °C. Sorting was performed with The Becton-Dickinson Influx™ cytometer (Becton Dickinson) using 640nm (Zombie NIR, 750LP filter), 355nm (calcein Blue AM, 460/50 filter), 488nm (CD45-PerCP, 692/40 filter) and 488nm (GFP, 530/40 filter) lasers. Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein Blue AM positive and Zombine NIR negative. We sorted viable CD45 negative/ GFP positive single-cells into 96-well plates containing 5uL of TCL buffer (Qiagen) with 1% beta-mercaptoethanol. Plates were frozen immediately after sorting and stored at −80°C prior to whole transcriptome amplification, library preparation and sequencing.

Neftel C., Laffy J., Filbin M.G., Hara T., Shore M.E., Rahme G.J., Richman A.R., Silverbush D., Shaw M.L., Hebert C.M., Dewitt J., Gritsch S., Perez E.M., Castro L.N., Lan X., Druck N., Rodman C., Dionne D., Kaplan A., Bertalan M.S., Small J., Pelton K., Becker S., Bonal D., Nguyen Q.D., Servis R.L., Fung J.M., Mylvaganam R., Mayr L., Gojo J., Haberler C., Geyeregger R., Czech T., Slavc I., Nahed B.V., Curry W.T., Carter B.S., Wakimoto H., Brastianos P.K., Batchelor T.T., Stemmer-Rachamimov A., Martinez-Lage M., Frosch M.P., Stamenkovic I., Riggi N., Rheinbay E., Monje M., Rozenblatt-Rosen O., Cahill D.P., Patel A.P., Hunter T., Verma I.M., Ligon K.L., Louis D.N., Regev A., Bernstein B.E., Tirosh I, & Suvà M.L. (2019). An integrative model of cellular states, plasticity and genetics for glioblastoma. Cell, 178(4), 835-849.e21.

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Multiparametric Flow Cytometry Analysis

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Expression of surface molecules was analyzed using the BD LSRFortessa (BD Biosciences, CA, USA) or the CYTEK Aurora (Cytek Biosciences, CA, United States). Data analysis followed the workflow in FlowJo (Tree Star, OR, United States). Cells were stained with diluted antibodies, and Zombie NIR (#423106, BioLegend) was used to distinguish live/dead cells. Staining was performed in wash buffer (BD Biosciences) for 30 min at 4 ^οC. For multiparametric assays, after exclusion of dead cell by Zombie NIR staining, memory T cells were gated based on expression of CD45RA and CCR7. Using FlowJo software, dimensionality reduction was performed applying uniform manifold approximation and projection (UMAP). FlowJo plugin Phenograph was used for the identification of unique cell populations (cluster) on the resulting UMAP map. Quantified frequencies of each cluster are presented as pie charts. To evaluate intracellular cytokine production, cells were treated with Cell Activation Cocktail (#423301, BioLegend) for 6 h, permeabilized using the BD Cytofix/Cytoperm Kit (#554714, BD Biosciences), stained with specific antibodies, and analyzed by flow cytometry. GolgiStop (#554724, BD Biosciences) was added at the same time to inhibit protein release to the extracellular space. The antibodies used in flow cytometry experiments are listed in Table S3.

Ohtsuki S., Wang C., Watanabe R., Zhang H., Akiyama M., Bois M.C., Maleszewski J.J., Warrington K.J., Berry G.J., Goronzy J.J, & Weyand C.M. (2023). Deficiency of the CD155-CD96 immune checkpoint controls IL-9 production in giant cell arteritis. Cell Reports Medicine, 4(4), 101012.

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Flow Cytometry Analysis of Splenocytes

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Mice that were immunized as described above were sacrificed 4 weeks after the third immunization. Spleens were collected into RPMI containing 2% fetal bovine serum and manually homogenized using the back of a syringe plunger. Cells were filtered through 75 um mesh, washed 1x, and counted. 2x10⁷ splenocytes were stained for flow cytometry. All washes for the staining process were performed in PBS containing 2% fetal bovine serum and 2 mM EDTA. Cells were incubated with CD16/32 (Biolegend Cat# 101302) and 5.875 μg/ml of biotinylated Abp2D_RBD for 10 minutes, then washed 3x. A cocktail containing the following antibodies was prepared in BD Brilliant Staining Buffer (BD Cat. # 563794), all sourced from BioLegend unless otherwise indicated : Zombie NIR (Cat#423105), CD19-BV750 (Cat#115561), CD4-BV570 (Cat#100542), IgD-BV711 (Cat#405731), IgM-BV605 (Cat#406523), IgG1-BV510 (Cat#406621), Fas-PE (BD Cat# 554258), GL7-PcpCy5.5 (Cat# 144610), CD38-PE-Cy7 (Cat#102718), CD138-BV421 (Cat#142508), and streptavidin-APC-Fire-750 (Cat#405250). Invitrogen UltraComp eBeads were used for single colors. Flow cytometry data was collected using the Cytek Aurora with 4 laser 16V-14B-10YG-8R configuration and processed on FlowJo10 for Mac.

Timm M.R., Tamadonfar K.O., Nye T.M., Pinkner J.S., Dodson K.W., Ellebedy A.H, & Hultgren S.J. (2023). Vaccination with Acinetobacter baumannii adhesin Abp2D provides protection against catheter-associated urinary tract infection. Research Square.

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