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Rm4 5

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The RM4-5 is a laboratory equipment designed for cell culture and analysis. It functions as a rotator that gently mixes and suspends cells or samples during incubation.

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Lab products found in correlation

Fjk 16, by Thermo Fisher Scientific (6 mentions) M1 70, by BioLegend (2 mentions) Golgiplug, by BD (2 mentions) Ifn γ xmg1.2, by BioLegend (2 mentions) Facsverse, by BD (2 mentions) Percp cy5.5 labelled anti cd11c n418, by BioLegend (2 mentions) Labeled anti nk 1.1 pk136, by Thermo Fisher Scientific (2 mentions) Fitc labeled anti ly6g rb6 8c5, by Thermo Fisher Scientific (2 mentions) Uniform dyed microspheres, by Bangs Laboratories (2 mentions) Fc block anti mouse cd16 32, by Thermo Fisher Scientific (2 mentions) Mel 14, by BioLegend (2 mentions) Facscanto flow cytometer, by BD (2 mentions) Anti mouse cd4, by BioLegend (1 mentions) Anti mouse gl7, by BioLegend (1 mentions) Anti mouse fas, by BioLegend (1 mentions) Apc cy7, by BioLegend (1 mentions) Pe efluor 610, by Thermo Fisher Scientific (1 mentions) Apc fire 750, by BioLegend (1 mentions) Apc efluor 780, by Thermo Fisher Scientific (1 mentions) H57 597, by BioLegend (1 mentions)

Spelling variants of this product

Anti-CD4 (RM4-5, (26 citations) FITC-anti-mouse CD4 (RM4-5, (2 citations) Anti-CD4-PerCP (clone RM4-5; (2 citations) FITC-anti-CD4 (clone RM4–5), (2 citations) Pacific blue-conjugated anti-CD4 (RM4-5); (2 citations) APC-CD4 RM4-5 (2 citations) CD4-PE-Cy7 clone RM4–5 (2 citations) Anti-mouse CD4 antibody conjugated to Biotin (clone RM4-5, (2 citations) Anti–mouse CD4 (clone RM4-5; (2 citations) Anti-CD4 BV711 (RM4-5; (2 citations)

29 protocols using rm4 5

1

Profiling Lymph Node Immune Cells

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The draining inguinal LNs were homogenized and filtered through a 70-μm cell strainer, and 2×106 cells were stained with the viability dye Near-IR and blocked with anti-mouse CD16/32 (S17011E clone, BioLegend, USA). Subsequently, the cells were stained with anti-mouse CD19 (Brilliant Violet 605™, 6D5 clone), anti-mouse GL7 (PE, GL7 clone), anti-mouse Fas (APC, SA367H8 clone), anti-mouse CD4 (FITC, RM4-5 clone), anti-mouse PD-1 (Brilliant Violet 421™, 29F.1A12 clone), anti-mouse CXCR5 (PE/Cyanine7, L138D7 clone) and anti-mouse CD3 (PerCP/Cyanine5.5, 17A2 clone), which were all purchased from BioLegend. After staining, the cells were fixed, resuspended in PBS, and then acquired on a BD FACS Canto™ flow cytometer.
Bian T., Wang B., Fu G., Hao M., Chen Y., Fang T., Liu S., Yu C., Li J, & Chen W. (2022). Single-dose of a replication-competent adenovirus-vectored vaccine provides sterilizing protection against Rift Valley fever virus challenge. Frontiers in Immunology, 13, 907675.
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2

Multiparameter Flow Cytometry Assay

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Antibodies against murine CD4 (RM4-5, AF700, BV650), CD8 (53-6.7, APC/Fire 750, BV785), CCR2 (SA203G11, BV421), CD11c (N418, APCCy7, Pe/Cy5), CD206 (C068C2, APC), CD11b (M1/70, BV421), F4/80 (BM8, BV510, BV711, FITC), TCRβ (H57-597, BV605), and NK1.1 (PK136, BV650) were purchased from BioLegend. Antibodies against CD4 (GK1.5, BUV395) and ST2 (U29-93, BV480) were purchased from BD Biosciences. Antibodies against B220 (RA3-6B2, AF488), CD11c (N418, e450, PE-Cy5.5), CD25 (PC61.5, PE-Cy7), CD4 (GK1.5, APC-eFluor 780, Super Bright 645), CD45 (30-F11, Pacific Orange), Foxp3 (FJK-16s, APC, e450, FITC), GITR (DTA-1, Super Bright 600), IL-10 (JES5-16E3, PE), KLRG1 (2F1, APC, APC-eFluor 780, PE-eFluor 610), NK1.1 (PK136, PE), and ST2 (RMST2-2, PE, PerCP-eFluor710) were purchased from eBioscience, Thermo Fisher Scientific. Viability dyes (e506, near IR, UV) were purchased from Invitrogen, Thermo Fisher Scientific.
Extracellular flow staining was performed in 2% FBS-PBS in the presence of BioLegend’s TruStain FcX for 30 minutes at 4°C. Fixation, permeabilization, and intracellular staining were performed using eBioscience’s (Thermo Fisher Scientific) Foxp3/transcription factor staining buffer set per manufacturer’s directions. Flow data were collected using a Cytek Aurora or BD Biosciences LSR and analyzed using FlowJo.
Beppu L.Y., Mooli R.G., Qu X., Marrero G.J., Finley C.A., Fooks A.N., Mullen Z.P., Frias AB J.r., Sipula I., Xie B., Helfrich K.E., Watkins S.C., Poholek A.C., Ramakrishnan S.K., Jurczak M.J, & D’Cruz L.M. (2021). Tregs facilitate obesity and insulin resistance via a Blimp-1/IL-10 axis. JCI Insight, 6(3), e140644.
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3

Germinal Center B Cell Isolation

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Draining lymph nodes were passed through 70 micron filters and resuspended in PBS supplemented with 1% FBS and 1mM EDTA. Cells were stained with anti-B220 (Biolegend, RA3-6B2), CD38 (Biolegend, Clone 90), T- and B-cell activation antigen (BD Biosciences, GL-7), CD138 (Biolegend, 281-2), as well as anti-CD4 (Biolegend, RM4-5) for exclusion gate, for 30 minutes at 4°C. Cell sort was performed on a MoFlo Astrios instrument (Dako-Cytomation). GC B cells were defined as CD4- B220+ CD138- CD38lo/- GL-7+.
Cavazzoni C.B., Hanson B.L., Podestà M.A., Bechu E.D., Clement R.L., Zhang H., Daccache J., Reyes-Robles T., Hett E.C., Vora K.A., Fadeyi O.O., Oslund R.C., Hazuda D.J, & Sage P.T. (2022). Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice. Cell Reports, 38(8), 110399.
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4

Mouse Blood Immunophenotyping with Flow Cytometry

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Heparin-treated peripheral mouse blood was treated with anti-CD16/32 antibody (2.4G2, BD Biosciences) to reduce nonspecific binding against mouse CD4 (RM4-5, Biolegend), CD8 (53–6.7, BD-Biosciences), CD11b (M1/70, BD Biosciences), CD45.1 (A20, Biolegend), CD45.2 (104, Biolegend), and Ly6C (HK1.4, Biolegend). Samples were analyzed using the Attune NxT Flow Cytometer equipped with 405 nm, 488 nm, 561 nm, and 640 nm lasers. Compensation was performed with isotype controls and single antibody-stained samples, and the data were analyzed using the FlowJo software (version 10.8.1).
Bian E.J., Chen C.W., Cheng C.M., Kuan C.Y, & Sun Y.Y. (2023). Impaired post-stroke collateral circulation in sickle cell anemia mice. Frontiers in Neurology, 14, 1215876.
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5

Intracellular Cytokine Analysis of Splenic and Ocular Cells

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Mouse spleen and eye infiltrating cell cultures were prepared and their intracellular expression of IL-17, IFN-γ, Τ−bet, or RORγt were determined as detailed elsewhere [23 , 26 (link)]. Briefly, the cells were incubated with ionomycin/PMA and Brefeldin A (GolgiPlug, BD Biosciences), followed by staining with conjugated anti-CD3 monoclonal antibody (145–2C11, BD Biosciences), anti-CD4 monoclonal antibody (RM4–5, Biolegend) and anti-CD45 monoclonal antibody (30-F11, Biolegend). After 4% paraformaldehyde fixation and permeabilization (BD bioscience) cells were intracellularly stained for T-bet (4B10, Biolegend), RORγt (AFKJS-9, BD Bioscience), IFN-γ (XMG1.2, Biolegend) and IL-17A (TC11–18H10.1, ebioscience). The staining patterns were analyzed by a MACSQuant Analyzer 10 (Miltenyi Bioitec. Cambridge, MA) and processed by FlowJo (Tree Star, Ashland, OR). Dead cells were excluded using ghost red 780 viability dye (Tonbo biosciences, San Diego, CA) and only single cells were gated for further analysis using FSC-A and FSC-H. Gating strategies are shown in supplementary Fig. S3 and Fig. S4.
Lyu C., Bing S.J., Wandu W.S., Xu B., Shi G., Hinshaw S.J., Lobera M., Caspi R.R., Lu L., Yang J, & Gery I. (2018). TMP778, a selective inhibitor of RORγt, suppresses experimental autoimmune uveitis development, but affects both Th17 and Th1 cell populations. European journal of immunology, 48(11), 1810-1816.
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6

Identification of mCMV-specific T-cells

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Cells were incubated with Fc block (mouse Fcγ III/II Receptor, 2.4G2, BD Pharmingen, San Jose, CA) for 15 min. Flourescent dye-conjugated antibodies specific for CD8 (53–6.7, APC), CD4 (RM4-5, PerCP/Cy5.5), and CD11c (N418, Alexa Fluor 700) were used (BioLegend, San Diego, CA). Alveolar macrophages were defined as CD11C+ and highly auto-fluorescent (Vermaelen 2004). mCMV-specific T-cells were identified using MHC-I tetramers specific for mCMV proteins (m123/pp89, H2Ld-restricted 168YPHFMPTNL176 and m164, H2Dd-restricted 257AGPPRYSRI265) (NIH Tetramer Core Facility, Emory University, Atlanta, GA) as previously described [Holtappels et al., 2002 (link); Sierro et al., 2005 (link)]. Briefly, MHC-I peptide tetrameric complexes were produced and assembled with PE-conjugated streptavidin. Cells were incubated with tetramers for 1 hr (37°C), followed by antibody surface staining for 30 min (4°C), fixed, and analyzed by flow cytometry (FACSAria III, Becton Dickinson, San Jose, CA), and results were analyzed using FlowJo software (Tree Star, Ashland, OR).
Mansfield S., Dwivedi V., Byrd S., Trgovcich J., Griessl M., Gutknecht M, & Cook C.H. (2016). Broncholaveolar Lavage to Detect Cytomegalovirus Infection, Latency, and Reactivation in Immune Competent Hosts. Journal of medical virology, 88(8), 1408-1416.
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7

Characterizing Neoantigen-Specific T Cell Responses

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Directly conjugated antibodies against murine CD3e (17A2), CD4 (RM4-5), CD8b (YTS156.7.7), IFN-γ (XMG1.2), TNF-α (MP6-XT22), and IL-2 (JES6-5H4) were purchased from BioLegend. A Live/Dead Violet viability kit (Invitrogen) was used to exclude dead cells from analysis. To determine intracellular cytokine production, 2 million splenocytes from vaccinated mice were cultured with peptides (5 μg/mL) derived from corresponding mutated neoantigen, protein transport inhibitor (eBioscience), and CD107a antibody (1D4B, BioLegend) for 5 h. Surface staining followed by intracellular cytokine staining was done to determine cytokine production. Cells were permeabilized using the eBioscience FoxP3 staining kit as per the manufacturer’s instructions. Data were acquired on a BD FACSymphony (BD Biosciences) and analyzed using FlowJo. The neoantigen peptides consisted of 15-mer peptides overlapping by 9 aa. The peptides were designed to cover the entire 33-mer used for vaccination. Mice were vaccinated three times at 2-week intervals and euthanized 1 week after the last vaccination. Spleens were harvested, and splenocyte suspensions were obtained using a Stomacher 80 Biomaster (Thomas Scientific), followed by red blood cell lysis (Thermo Fisher Scientific).
Bhojnagarwala P.S., Perales-Puchalt A., Cooch N., Sardesai N.Y, & Weiner D.B. (2021). A synDNA vaccine delivering neoAg collections controls heterogenous, multifocal murine lung and ovarian tumors via robust T cell generation. Molecular Therapy Oncolytics, 21, 278-287.
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8

Comprehensive Immune Cell Profiling

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Anti-mouse phosphor-STAT3 (EP2147Y, Abcam), phosphor-NF-κB (pp65) (sc-52401, Santa), phosphor-JNK (pJNK)(9H8), phosphor-ERK (pERK)(197G2), STAT3 (124H6, Cell Signaling), NF-κBp65 (sc8008, Santa), JNK (SC), ERK(137F5), and β-actin (sc-47778, Santa) were purchased. FITC-, PE-, APC- APC/cy7-, PerCP/Cy5.5-, or PE/cy7-conjugated anti-mouse CD4 (RM4-5, Biolegend), CD8 (ZUT270.5, Biolegend), CD45.1 (30-F11, Biolegend), MHCII (I-A/I-E, M5/114.115.2, Biolegend), CD11c (MCA1441GA, Biolegend), CD11b (M1/70, eBioscience), F4/80 (BM8, Biolegend), CD206 (CO68C2), Gr-1(RB6-8C5, eBioscience), IL-10 (JES5-16E3, eBioscience), Foxp3 (MF23, eBioscience), IL-22 (140301, R&D system), IFNγ (XMG1.2, Biolegend), RORγt (AFKJS-9, eBioscience), IL-17A (eBio17B7, eBioscience), and CD3ζ (H146-968, Abcam) antibodies were purchased. Anti- REG3γ (PA517, Thermo) and anti-MUC2 (H300, Santa) were also purchased. FITC-, PE-, APC- APC/cy7-, PerCP/Cy5.5-, or PE/cy7-conjugated isotypic controls were from Biolegend. RPMI1640, DMEM, fetal bovine serum (FBS), and antibiotics were obtained from HyClone. Alexa fluor 488-conjugated goat anti-rabbit IgGH&L and Alexa fluor 594-conjugated goat anti-rabbit IgGH&L (Abcam) were also purchased. 7-AAD was from Abcam.
Lactobacillus reuteri (ATCC PTA 4659) was a gift of BioGaaia, Sweden.
Huang Y., Qi H., Zhang Z., Wang E., Yun H., Yan H., Su X., Liu Y., Tang Z., Gao Y., Shang W., Zhou J., Wang T., Che Y., Zhang Y, & Yang R. (2017). Gut REG3γ-Associated Lactobacillus Induces Anti-inflammatory Macrophages to Maintain Adipose Tissue Homeostasis. Frontiers in Immunology, 8, 1063.
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9

Cell Cycle Analysis of Activated T Cells

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mTh1 cells (0.5-1x106) were plated on anti-CD3/CD28 coated 48-well plates for 48h in the presence of DMSO or 2.5µM HLCL65. Cells were incubated with 10µM BrdU for the last 4 h at 37°C (BrdU APC flow kit, BD 552598). For controls, 3µM aphidocolin for G1/S arrest or 0.25µM paclitaxel for G2/M arrest were added 15 min prior to addition of BrdU. Cells were stained as described in manufacturer’s instructions. Briefly, cells were blocked 10 min at 4°C in FC block (Biolegend). Primary antibodies, CD4-e450 (48-0042-82, eBioscience clone RM4-5) and CD44-FITC (103006, Biolegend, clone IM7) were added and incubated 15 min at 4°C. Cells were washed, fixed with BD Fixation Solution for 20 min at 4°C, then washed with BD Perm Wash and stored overnight in FACS. The next day, they were washed in BD Perm Wash, incubated in BD CytoPerm Permeabilization Buffer Plus 10 min at 4°C, refixed with BD Fixation solution, 5 min at 4°C, incubated with DNase solution 1h at 37°C, washed and stained with BrdU 20 min at RT, washed and resuspended with 7-AAD to stain total DNA. Cells were then run on a FACSCanto flow cytometer (BD) and analyzed by FlowJo software.
Amici S.A., Osman W, & Guerau-de-Arellano M. (2021). PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity. Frontiers in Immunology, 12, 695947.
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10

Confocal Microscopy for Immune Cell Phenotyping

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Confocal microscopy was performed as described previously (26 (link)). Briefly, tissues were embedded in OCT medium (Tissue Tek) and 12 μm sections were cut using a cryostat. Sections were fixed and stained using the FoxP3 buffer set (eBiosciences) and directly conjugated antibodies. Sections were imaged on an Olympus confocal microscope with a 20x objective. For micrograph panels, single z slices were linearly contrasted and merged images were made in Adobe Photoshop. The following directly conjugated antibodies were used: anti-CD8a (53-6.7, BioLegend), anti-FoxP3 (FJK-16s, eBioscience), and anti-CD4 (RM4-5, BioLegend).
Cooper Z.A., Juneja V.R., Sage P.T., Frederick D.T., Piris A., Mitra D., Lo J.A., Hodi F.S., Freeman G.J., Bosenberg M.W., McMahon M., Flaherty K.T., Fisher D.E., Sharpe A.H, & Wargo J.A. (2014). Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade. Cancer immunology research, 2(7), 643-654.
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