Rat serum

Manufactured by Jackson ImmunoResearch

Sourced in United States

Rat serum is a biological fluid obtained from the blood of rats. It contains a complex mixture of proteins, hormones, and other molecules that are essential for various physiological processes. Rat serum is commonly used in research applications as a supplement to cell culture media, providing necessary nutrients and growth factors to support the proliferation and maintenance of cultured cells.

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Lab products found in correlation

Cell dissociation buffer, by Merck Group (1 mentions) Facsdiva software, by BD (1 mentions) Pacific orange, by Thermo Fisher Scientific (1 mentions) Cd62l mel 14, by BioLegend (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Normal mouse serum, by Jackson ImmunoResearch (1 mentions) Cd138 pe 281 2, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Facscanto 2, by BD (1 mentions) Fc block, by BD (1 mentions) Streptavidin apc, by BD (1 mentions) Pe conjugated anti mouse iga clone ma 6e1, by Thermo Fisher Scientific (1 mentions) Heparin, by Merck Group (1 mentions) Percoll plus, by GE Healthcare (1 mentions) Anti rabbit pe, by Jackson ImmunoResearch (1 mentions) Streptavidin pe, by BioLegend (1 mentions) Anti human igg biotin, by Jackson ImmunoResearch (1 mentions) Macs mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions) Foxp3 fix perm solution, by BioLegend (1 mentions) Foxp3 perm buffer, by BioLegend (1 mentions)

9 protocols using rat serum

BV2 Cell Characterization by Flow Cytometry

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BV2 cells were plated (3×10⁵ cells/well) in 24-well culture plates in DMEM containing 10% (v/v) FBS and 4.5 g/L D-glucose. Cells were allowed to grow overnight at 37°C with 5% CO₂ before replacing medium with DMEM-High or -Low (n = 5 for each group). After a 24-h incubation, cells were harvested upon treatment with cell dissociation buffer (Sigma-Aldrich) and transferred to 5-mL FACS tubes (BD Biosciences). Cells were spun down for 10 min at 300 x g and resuspended in DPBS containing 4% rat serum (Jackson ImmunoResearch). Cell metabolism was stopped by storage for 20 min on ice, and cells were then collected by centrifugation, washed and further incubated in 0.5 g/L Fc Block CD16/CD32 on ice for 20 min. Cells were next washed once with DPBS and suspended in 100 μL of DPBS containing 0.5 g/L Alexa 647-conjugated anti-CD68 antibody (AbD Serotec), and incubated on ice for 30 min. After one rinsing with DPBS, cells were resuspended in DPBS. Data were acquired using a flow cytometer (at least 10,000 singlet events) and analyzed with BD FACS Diva software.

Bordeleau M., ElAli A, & Rivest S. (2016). Severe chronic cerebral hypoperfusion induces microglial dysfunction leading to memory loss in APPswe/PS1 mice. Oncotarget, 7(11), 11864-11880.

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Flow Cytometry Analysis of Immune Cells

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Single cell suspensions were analyzed with flow cytometry. First, unspecific antibody staining was reduced by incubation with CD16/32 block (TueStain fcX™, BioLegend, San Diego, CA) and rat serum (Jackson Immuno Research, Bar Harbor, ME). Monoclonal antibodies specific for CD3 (clone 145-2C11), CD8 (53-6.7), CD4 (RM4-5), CD44 (IM7) and CD62L (MEL-14) were purchased from BioLegend and eBioscience. Pacific Orange (Life Technologies, Carlsbad, CA) was used for discrimination of dead cells. For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience/ThermoFischer Scientific, Waltham, MA) following the manufacturer’s instructions. Subsequently, staining of the transcription factor Foxp3 (FJK-16s, eBioscience/ThermoFischer Scientific, Waltham, MA) was performed. After washing, cells were reconstituted in 2% BSA/2 mM EDTA PBS before multicolor acquisition at the LSR II flow cytometer (BD Bioscience, Heidelberg, Germany). For each condition 2-12 biological replicates were measured in duplicates. Data analysis was done using FlowJo software (Tree Star, Ashland, OR).

Hierweger A.M., Engler J.B., Friese M.A., Reichardt H.M., Lydon J., DeMayo F., Mittrücker H.W, & Arck P.C. (2019). Progesterone modulates the T cell response via glucocorticoid receptor-dependent pathways. American journal of reproductive immunology (New York, N.Y. : 1989), 81(2), e13084.

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Single Cell Sorting of Antigen-Specific B Cells

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The isolated leukocytes were labeled with biotinylated HA, followed by streptavidin-APC (BD Biosciences). In addition, the following surface markers were used: CD3e-V450 (500A2), CD4-V450 (RM4-5), CD8a-V450 (53-6.7), CD11b-V450 (M1/70), IgD-V450 (11-26c.2a), Gr1-V450 (RB6-8C5), CD19-PE-Cy7 (1D3), CD138-PE (281-2) and IgM-FITC (R6-60.2) (BD Biosciences, San Jose, CA). All staining was performed in the following antibody dilution buffer: Hank’s Balanced Salt Solution (HBSS) containing 5 % FBS (Hyclone), 10 mM of EDTA, 10mM of HEPES, 1% Fc Block (BD Biosciences, San Jose, CA) and 5% each of normal hamster serum, normal mouse serum and normal rat serum (Jackson ImmunoResearch). The optimal staining conditions identified were 5 μg/ml of biotinylated-HA and 1.25 μg/ml of streptavidin-APC.
Cells were analyzed using a BD FACS CANTO II, and single cell sorting was performed using a BD FACS Aria II. The cells were gated on FSC-A/FSC-H to remove doublets and FSC-A/SSC-A to remove debris. Single antigen-positive B cells were sorted based on IgM-/CD19+/HA+ surface staining onto the 48 reaction sites of an AmpliGrid AG480F slide (Beckman Coulter, Houston, TX). A visual analysis of cell deposition on the AmpliGrid AG480F slide using a microscope was used to monitor the accuracy of the sort.

Wilson J.R., Tzeng W.P., Spesock A., Music N., Guo Z., Barrington R., Stevens J., Donis R.O., Katz J.M, & York I.A. (2014). Diversity of the murine antibody response targeting influenza A(H1N1pdm09) hemagglutinin. Virology, 458-459, 114-124.

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Quantification of Intestinal IgA by Flow Cytometry

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Faeces were collected and weighed, and then suspended in PBS to 100 mg ml⁻¹ by vigorous vortexing at 4 °C for 30 min. Each 500-μl suspension was centrifuged at 4 °C, 13,000 r.p.m. for 10 min. The pellet was then suspended in 1 ml 4% paraformaldehyde and incubated at 4 °C overnight with rotation. Each suspension was centrifuged at 4 °C, 1,000 r.p.m. for 2 min, and then 5 μl of the supernatant was washed twice with FACS buffer (2% FBS in PBS). Pellets were stained with FACS buffer containing 1 mg ml⁻¹ rat serum (Jackson ImmunoResearch) and 1 μg ml⁻¹ PE-conjugated anti-mouse IgA (clone mA-6E1, eBioscience) at 4 °C for 20 min, and were then analysed on NovoCyte Flow Cytometer Systems (ACEA). IgA-positive gate was determined by using isotype control antibody (Rat IgG1 κ, clone: RTK2071, BioLegend; Supplementary Fig. 7).

Kishikawa S., Sato S., Kaneto S., Uchino S., Kohsaka S., Nakamura S, & Kiyono H. (2017). Allograft inflammatory factor 1 is a regulator of transcytosis in M cells. Nature Communications, 8, 14509.

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Stromal Cell Enrichment Protocol

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Enzymatically digested thymus cell suspensions were enriched for stromal cells by density centrifugation. Briefly, 3 ml single-cell suspension in PBS was underlaid with 57% Percoll PLUS (GE) in PBS and spun for 20 min at 1800 rpm at 4 °C without brake. The cells at the interface were collected and transferred into a new tube, washed with PBS, and used for staining. Typically, 10⁶ cells were used in a single test. First, the cells were incubated with 6 U/ml Heparin (Sigma) for 10 min in ice in PBS to remove all HS-bound proteins, or with 100 μl of a mixture of B. eggerthii HSases (NEB) at 0.5 U/ml each in PBS for 1 h at 37˚C to digest HS, or with PBS only. After washing with FACS buffer, the cells were treated with 4 μg/ml recombinant CCL21 or 10 μg/ml of the different Fc fusion proteins for 1 h on ice. CCL21 was detected with anti-CCL21 antibody (LifeSpan) followed by anti-rabbit-PE (Jackson Immunoresearch). The Fc fusion proteins were detected with anti-human IgG-biotin (Jackson Immunoresearch) preincubated with 2% rat serum (Jackson Immunoresearch) followed by Streptavidin-PE (BioLegend). The cells were also stained for HS (10E4), CD45, EpCAM, and gp38, as described in the Flow Cytometry section.

Hsu H.P., Chen Y.T., Chen Y.Y., Lin C.Y., Chen P.Y., Liao S.Y., Lim C.C., Yamaguchi Y., Hsu C.L, & Dzhagalov I.L. (2021). Heparan sulfate is essential for thymus growth. The Journal of Biological Chemistry, 296, 100419.

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Prostate Tissue Dissociation and Myeloid Cell Analysis

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Prostate tissue was subjected to single cell dissociation using the MACS Mouse Tumor Dissociation Kit protocol and gentleMACS Dissociator (Miltenyi). Suspended cells were blocked with rat serum (012–000-120, Jackson ImmunoResearch), stained with FVS570 viability dye (1 ul/ml, 564995, BD Biosciences) in the dark for 15 minutes at room temperature. Samples were washed with PBS and incubated with Myeloid extracellular antibody panel (Supplementary Table S1) or corresponding isotype panels diluted in Brilliant Stain Buffer (566349, BD Biosciences) in the dark for 30 minutes at 4°C. Cells were washed with FACS buffer (1x PBS, 1% BSA, 2mM EDTA), fixed with 1x Fixation Buffer (420801, BioLegend) in the dark for 20 minutes at room temperature, and stored overnight in FACS buffer at 4°C. Samples were incubated in 1x FoxP3 Fix/Perm Solution (421401, BioLegend) in the dark for 20 minutes at room temperature and washed with 1x FoxP3 Perm Buffer (421402, BioLegend). Cells were resuspended with Myeloid intracellular antibody panel (Supplementary Table S1) or corresponding isotype panels diluted in FACS buffer in the dark for 30 minutes at room temperature under gentle agitation. Cell suspensions were washed with FACS buffer and analyzed with a Gallios flow cytometer (Beckman Coulter Life Sciences). Macrophages were defined as FVS570⁻CD45⁺CD11b⁺F4/80⁺CD68⁺ cells.

de Groot A.E., Myers K.V., Krueger T.E., Kiemen A.L., Nagy N.H., Brame A., Torres V.E., Zhang Z., Trabzonlu L., Brennen W.N., Wirtz D., De Marzo A.M., Amend S.R, & Pienta K.J. (2021). Characterization of tumor-associated macrophages in prostate cancer transgenic mouse models. The Prostate, 81(10), 629-647.

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Flow Cytometry of Corneal Leukocytes

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Flow cytometry was performed as previously described (Robinson et al., 2013b (link); Zhou et al., 2012 (link)). Corneas were dissected from mouse eyes at one day post infection and digested with 1 mg/mL collagenase type I and 0.5 mg/mL DNase (Sigma Chemical Co., St. Louis, MO). Single cell suspensions were washed in phosphate buffered saline (PBS) and incubated on ice for 15 minutes with 2 μl anti-mouse Fc block (BD Pharmingen, San Diego, CA) in a total volume of 100 μl PBS-1% bovine serum albumin. After incubation, cells were centrifuged (300 x g for 5 minutes) and resuspended in 5% rat serum (Jackson Immuno Research Inc., West Grove, PA) for an additional 15 minutes on ice. Cells were then labeled with 4 μl anti-mouse FITC-conjugated anti-CD45 (clone 30-F11, BD Pharmingen), and incubated in the dark on ice for 30 minutes. Then, the cells were washed with PBS-1% BSA and resuspended in PBS containing 1% paraformaldehyde. After overnight fixation at 4°C in the dark, cells were pelleted, resuspended in PBS-1% BSA. Flow cytometry was performed using a Cytomics FC500 (Beckman Coulter, Brea, CA) for CD45+ events, representing the number of fluorescent events per cornea, roughly corresponding to the numbers of leukocytes.

Singh G., Zhou X., Lee J.Y., Yousuf M.A., Ramke M., Ismail M.A., Lee J.S., Robinson C.M., Seto D., Dyer D.W., Jones M.S., Rajaiya J, & Chodosh J. (2015). Recombination of the Epsilon Determinant and Corneal Tropism: Human Adenovirus Species D Types 15, 29, 56, and 69. Virology, 485, 452-459.

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Multimodal Profiling of Lymphoma Cells

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Global gene expression profiling (GEP) relied on Illumina Mouse WG-6 v2.0 (San Diego, CA, USA) Bead Chips and data analytical approaches described elsewhere.^{32 (link)} NFκB DNA-binding activity was determined with the help of EMSA.^{33 (link)} Western blotting followed a recently published protocol^{34 (link)} using antibodies listed in Supplementary Material and Methods. Capillary-based Sanger sequencing of Myd88^L252 and flanking regions relied on an ABI 3730xl (Thermo Fisher Scientific, Foster City, CA, USA) instrument provided by the HCCC Genomics Core. FISH was performed on metaphase chromosomes using gene-specific probes for Igh and Myc.^{23 (link)} Images were acquired using a DMRXA epifluorescence microscope equipped with a Sensys charge-coupled device camera (Roper Scientific, Trenton, NJ, USA). Surface expression of B cell and plasma cell markers was analyzed with the help of a FACSCanto II flow cytometer (Becton Dickinson (BD), San Jose, CA, USA)^{35 (link)} and antibodies in Supplementary Material and Methods. Non-specific Ab binding was blocked using rat serum (Jackson Immunoresearch, West Grove, PA, USA) and 10 μg 2.4G2 (BioXCell, West Lebanon, NH, USA). Data were analyzed using FlowJo (Tree Star, Ashland, OR, USA).

Tompkins V.S., Sompallae R., Rosean T.R., Walsh S., Acevedo M., Kovalchuk A.L., Han S.S., Jing X., Holman C., Rehg J.E., Herms S., Sunderland J.S., Morse H.C, & Janz S. (2016). Transgenic mouse model of IgM+ lymphoproliferative disease mimicking Waldenström macroglobulinemia. Blood Cancer Journal, 6(11), e488-.

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Multicolor flow cytometry for immune cell profiling

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Freshly prepared cell suspensions were blocked with 1% mouse serum (Jackson ImmunoResearch), 1% rat serum (Jackson ImmunoResearch), and 2% mouse FcR Blocking Reagent (Miltenyl) for 15 mins at 4 o C. Fluorophore-conjugated primary antibodies against cell surface antigens were added to the cell suspensions and incubated for 30 mins at 4 o C. Cells were then washed with cold PBS and stained with LIVE/DEAD UV (Invitrogen) according to manufacturer's instructions. Cells were then washed with cold FACS buffer and fixed with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to manufacturer's protocol. Fixed cells were again blocked with 1% mouse serum, 1% rat serum, and 2% mouse FcR Blocking Reagent for 15 mins at 4 o C and then stained with fluorophore-conjugated primary antibodies against intracellular antigens. Cells were then washed, and data were acquired on a LSR II Flow Analyzer (BD). Details on the antibodies used are provided in Supplementary Table S4. Data acquired were analyzed by using FlowJo software (BD).

Tang K.H., Li S., Khodadadi‐Jamayran A., Jen C.J., Han H., Guidry K., Chen T., Yuan H., Fedele C., Zebala J.A., Maeda D.Y., Christensen J.G., Olson P., Athanas A., Wong K., & Neel B.G. (2021). Combined Inhibition of SHP2 and CXCR1/2 Promotes Anti-Tumor T Cell Response in NSCLC.

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