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Cd4 clone gk1

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CD4 (clone GK1.5) is a mouse monoclonal antibody that binds to the CD4 surface antigen. CD4 is a co-receptor expressed on the surface of T helper cells, monocytes, macrophages, and dendritic cells. It plays a role in the activation and differentiation of these cell types.

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Lab products found in correlation

Cd8 clone 53 6, by BioLegend (15 mentions) Cd11b clone m1 70, by BioLegend (9 mentions) Cd3 clone 17a2, by BioLegend (8 mentions) Cd11c clone n418, by BioLegend (8 mentions) Thy1.2 clone 30 h12, by BioLegend (8 mentions) Thy1.1 clone ox 7, by BioLegend (8 mentions) Cd8a clone 53 6, by BioLegend (6 mentions) Cd44 clone im7, by BioLegend (6 mentions) Cd19 clone 6d5, by BioLegend (5 mentions) Cd45 clone 30 f11, by BioLegend (5 mentions) Ly6c clone hk1, by BioLegend (5 mentions) Cd3e clone 145 2c11, by BioLegend (4 mentions) Lsr 2 flow cytometer, by BD (4 mentions) Cd62l clone mel 14, by BioLegend (4 mentions) Facscanto 2, by BD (3 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (3 mentions) Nk1.1 clone pk136, by BioLegend (3 mentions) Clone n418, by BioLegend (3 mentions) Lsrii analyser, by BD (3 mentions) Cd95 clone jo2, by BD (3 mentions)

Spelling variants of this product

CD4 (GK1.5, (44 citations) Clone GK1.5 (14 citations) CD4 APC/Cy7 (clone GK1.5 (7 citations) FITC-CD4 (clone GK1.5). (2 citations) α‐CD4 (clone GK1.5, (2 citations) Anti‐CD4 antibody (clone GK1.5, (2 citations) Anti-CD4-BV421 (clone GK1.5; (2 citations) Anti-CD4-PerCp5 (clone Gk1.5, (2 citations) PE-Cy7 anti-mouse CD4 (clone GK1.5), (2 citations) Rat anti-CD4 (Clone GK1.5) (1 citations)

42 protocols using cd4 clone gk1

1

Flow Cytometry Analysis of Immune Cell Subsets

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To detect surface proteins, mononuclear cells were incubated with Fc Block (Bio X Cell, 2.4G2) for 15 min and washed, followed by incubation with viability dye. The indicated antibodies were fluorescently conjugated against CD45 (clone 30-F11, BD Horizon, cat #563410), CD11b (clone M1/70, BD Biosciences, cat #563553), CD11c (clone HL3, BD Pharmingen, cat #553801), CD4 (clone GK1.5, BioLegend, cat #100428), CD8α (clone 53 – 6.7, BioLegend, cat #100734), CD19 (clone 6D5, BioLegend, cat #115541), and MHC Class II (clone M5/114.15.2, BioLegend, cat #107628). Samples were run on a BD Symphony (BD Biosciences) and analyzed using FlowJo software (Tree Star), as described (31 (link), 40 (link), 41 (link)).
Hong H., Wang Y., Menard M., Buckley J., Zhou L., Volpicelli-Daley L., Standaert D., Qin H, & Benveniste E. (2024). Suppression of the JAK/STAT Pathway Inhibits Neuroinflammation in the Line 61-PFF Mouse Model of Parkinson’s Disease. Research Square.
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2

Multiparametric Immune Phenotyping of Cells

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Single cell suspensions were treated with Zombie violet as per the manufacturers insert instructions (Biolegend). Following treatment with Fc block (1 μg/ml) (Biolegend), cells were stained with combinations of the following antibodies: CD69 (clone H1.2F3), CD44 (clone IM7), CD8α (clone 53–6.7), CD3e (clone 145– 2C11), CD3 (clone 17A2), CD11a (clone I21/7), CD45 (clone 30-F11), Thy1.2 (clone 53–2.1), CD4 (clone GK1.5), CD4 (clone RM 4–4), CD4 (clone RM 4–5), CD62L (clone MEL-14) (BioLegend). Intracellular cytokine staining was performed with anti-IFN-γ (clone XMG1.2) and anti-IL17A (clone TC11–18H10.1) antibodies (BioLegend). Data was acquired using a FACS Canto II flow cytometer (Becton Dickinson (BD)) or Attune NxT flow cytometer (ThermoFischer Scientific) and analyzed using FlowJo software (TreeStar, version 10). All gating stragies used are described in the supplementary data.
O’Hara J.M., Redhu N.S., Cheung E., Robertson N.G., Patik I., Sayed S.E., Thompson C.M., Herd M., Lucas K.B., Conaway E., Morton C.C., Farber D.L., Malley R, & Horwitz B.H. (2019). Generation of protective pneumococcal-specific nasal resident memory CD4+ T cells via parenteral immunization. Mucosal Immunology, 13(1), 172-182.
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3

Isolation and Analysis of Midbrain Mononuclear Cells

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Mononuclear cells were isolated 4 weeks post-transduction from ventral midbrains with bilateral AAV injections, according to published protocols [27 (link), 30 (link)]. Briefly, midbrains were digested with 1 mg/mL Collagenase IV (Sigma) and 20 μg/mL DNAse I (Sigma) diluted in RPMI 1640 with 10% heat inactivated fetal bovine serum, 1% L-glutamine (Sigma), and 1% Penicillin-Streptomycin (Sigma). Mononuclear cells were separated out using a 30/70% Percoll gradient, as previously described [30 (link)]. Isolated cells were blocked with anti-Fcy receptor (clone 2.4G2 BD Biosciences) then incubated with fluorescent-conjugated antibodies against CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, BioLegend), MHCII (M5/114.15.2, BioLegend), Ly6C (clone HK 1.4, BioLegend), CD4 (clone GK1.5, BioLegend), and CD8a (clone 53-6.7, BioLegend). A fixable viability dye was used to distinguish live cells from debris per manufacturer’s instructions (Fixable Near-IR LIVE/DEAD Stain Kit, Invitrogen). Samples were analyzed using an Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo software (Tree Star).
Williams G.P., Schonhoff A.M., Jurkuvenaite A., Thome A.D., Standaert D.G, & Harms A.S. (2018). Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson’s disease. Journal of Neuroinflammation, 15, 244.
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4

Identification of Lung ILC2 Cells

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Lung ILC2 cell identification was performed as described previously (47 (link)). Lung tissues were digested in 8 mL RPMI 1640 containing liberase (50 μg/mL) and DNase I (1 μg/mL) for approximately 40 minutes at 37°C. Cell suspensions were filtered through 70 μm cell strainers and washed once with RPMI 1640. For ILC2 cell identification, total lung cell suspensions were blocked with 2.4G2 antibodies and stained with lineage cocktail mAbs: CD3ε (clone 145-2C11) (BioLegend, 100304), CD4 (clone GK1.5) (BioLegend, 100404), CD8α (clone 53-6.7) (Tonbo Biosciences, 30-0081-U500), CD11c (clone N418) (BioLegend, 117304), FceRIα (clone MAR-1) (BioLegend, 134304), NK1.1 (clone PK136) (BioLegend, 108704), CD19 (clone 6D5) (BioLegend, 115504), TER119 (clone TER-119) (BioLegend, 116204), CD5 (clone 53-7.3) (BioLegend, 100604), F4/80 (clone BM8.1) (Tonbo Biosciences, 30-4801-U500), Ly6G (clone RB6-8C5) (Tonbo Biosciences, 30-5931-U500), APC-conjugated streptavidin (BioLegend, 405207), PE-conjugated T1/ST2 (clone DIH9) (BioLegend, 145304), PerCP-Cy5.5-conjugated CD25 (clone PC61) (BioLegend, 102030), V450-conjugated Sca-1 (clone D7) (BD Biosciences, 560653), PE-Cy7-conjugated KLRG1 (clone 2F1/KLRG1) (BioLegend, 138416), APC-Cy7-conjugated CD45, and Fixable Viability Dye eFluor 506.
She L., Barrera G.D., Yan L., Alanazi H.H., Brooks E.G., Dube P.H., Sun Y., Zan H., Chupp D.P., Zhang N., Zhang X., Liu Y, & Li X.D. (2021). STING activation in alveolar macrophages and group 2 innate lymphoid cells suppresses IL-33–driven type 2 immunopathology. JCI Insight, 6(3), e143509.
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5

Immune Cell Profiling in Murine Super-Infection

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Whole blood was collected from mice on days −1, 3, 7 and 9 post‐super‐infection. Blood was collected in 0.5 M EDTA, then lysed using ACK Lysis buffer, washed and resuspended in flow cytometry buffer (2% FCS + 0.5 mM EDTA in PBS). Single‐cell suspensions were blocked with anti‐CD16/32 antibody and stained with fluorochrome‐conjugated antibodies against mouse CD3 (clone 17A2; Biolegend, San Diego, CA, USA), CD4 (clone GK1.5, Biolegend), CD8 (clone 53‐6.2, BD Biosciences, Franklin Lakes, NJ, USA), CD45 (clone 30‐F11, BD Biosciences), CD11b (clone M1/70, BD Biosciences), IA/IE (M5/114; eBioscience, San Diego, CA, USA), Ly6G (clone 1A8, BD Biosciences), CD62L (clone MEL‐14, BD Biosciences) and CD69 (clone H1.2F3, BD Biosciences), and dead cells were excluded using LIVE/DEAD Near‐Infrared viability dye (#L110909, Thermo Fisher Scientific). Sphero™ Blank Calibration Particles (BD Biosciences) were added to the samples and used as reference counting beads. Cells were acquired on a BD LSR II Fortessa Cell Analyser, and data were analysed using FlowJo software (v10.6; Treestar, Inc.).
Zaman M., Huber V.C., Heiden D.L., DeHaan K.N., Chandra S., Erickson D., Ozberk V., Pandey M., Bailly B., Martin G., Langshaw E.L., Zaid A., von Itzstein M, & Good M.F. (2021). Combinatorial liposomal peptide vaccine induces IgA and confers protection against influenza virus and bacterial super‐infection. Clinical & Translational Immunology, 10(9), e1337.
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6

Induction of Regulatory T Cells by CVF- and CLys-treated BMDCs

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BMDCs were generated as above and incubated for 72 h with CVF or CLys. 24 h before the end of culture, cells were pulsed overnight with endotoxin‐free OVA protein (20 μg/ml). On day 0, CVF‐ and CLys‐treated BMDCs were extensively washed with PBS, and 2 × 105 antigen‐pulsed cells, or PBS or naive BMDCs as control, were subcutaneously injected into separate recipient C57BL/6 mice, which had previously (24 h earlier) received intraperitoneally 1–2 × 105 flow sorted naïve CD4+CD44−/lo OT‐II cells. Mice were sacrificed on day 7, the inguinal lymph nodes (ILN) were removed and ILN cell suspensions, prepared as previously reported (Layland et al, 2010 (link)), were stained with CD4 (clone GK 1.5) (BioLegend), CD45.1 (clone A20), CD25 (clone PC61), FoxP3 (clone MF23) (all from BD Biosciences), and Granzyme B (clone NGZB) (eBioscience) for flow cytometry analysis of Treg induction.
Prodjinotho U.F., Gres V., Henkel F., Lacorcia M., Dandl R., Haslbeck M., Schmidt V., Winkler A.S., Sikasunge C., Jakobsson P., Henneke P., Esser‐von Bieren J, & Prazeres da Costa C. (2022). Helminthic dehydrogenase drives PGE2 and IL‐10 production in monocytes to potentiate Treg induction. EMBO Reports, 23(5), e54096.
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7

Comprehensive Immune Cell Phenotyping

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Cell suspensions were stained in ice cold PBS with or without 2 % FBS on ice. The following antibodies were used for staining mouse cells: CD45 (clone 30-F11, Biolegend), Ly6G (clone 1A8, BD Biosciences), CD11c (clone HL3, BD Biosciences), CD11b (clone M1/70, Biolegend), Siglec F (clone E50–2440, BD Biosciences), CD3e (clone 145–2C11, eBioscience), CD4 (clone GK1.5, Biolegend), IFN © (clone XMG1.2, Biolegend), IL-17 (clone eBio17B7, eBioscience), IL-5 (clone TRFK5, Biolegend), IL-13 (clone eBio13A, eBioscience), CD25 (clone PC61.5, eBioscience), CD44, CD62L (clone MEL-14, BD Biosciences), MHC class II I-A/I-E (clone M5/114.15.2, eBioscience), DC-SIGN/CD209 (R&D systems), CD32 (clone D34–485, eBioscience), TLR4/CD284 (clone SA15–21, Biolegend), Annexin V (Biolegend). Neutrophils were defined as CD45+CD11b+Ly6G+DNA+ and cytoplasts were defined at CD45+CD11b+Ly6G+DNA−. Naïve T lymphocytes were defined as CD4+CD25−CD44lowCD62Lhi. Lung Dendritic cells were defined as CD45+CD11c+ MHCII+autofluorescencelow. The following antibodies were used to stain human cells: anti-CD45 PE-Cy7 (HI30), anti-CD66b (G10F5), anti-CD16 APC-Cy7 (3G8) all from Biolegend. Vybrant DyeCycle Ruby (Life Technologies) was used to stain intracellular DNA. Data were acquired on BD Canto II or BD LSR Fortessa and analyzed using FlowJo v10. For cell sorting, FACS Aria was used.
Krishnamoorthy N., Douda D.N., Brüggemann T.R., Ricklefs I., Duvall M.G., Abdulnour R.E., Martinod K., Tavares L., Wang X., Cernadas M., Israel E., Mauger D.T., Bleecker E.R., Castro M., Erzurum S.C., Gaston B.M., Jarjour N.N., Wenzel S., Dunican E., Fahy J.V., Irimia D., Wagner D.D, & Levy B.D. (2018). Neutrophil cytoplasts induce Th17 differentiation and skew inflammation toward neutrophilia in severe asthma. Science immunology, 3(26), eaao4747.
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8

Tracking T Cell Responses to BMDC Transplant

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Thy1.1+/1.2+ P14 T cells and Thy1.1+/1.1+ SMARTA T cells were isolated from TCR Tg mice as detailed above. 105 P14 T cells and 105 SMARTA T cells were co-transferred I.V. by retro-orbital injection into wild-type B6 recipients 0–19 days prior to BMDC transplant.
BMDCs were prepared from the bone marrow of NINJA mice as described in47 (link). Four days after bone marrow harvest, maturing BMDCs were transduced in vitro with 1010 PFU/mL of recombinant Ad5CMVFLPo (Iowa Vector Core, VVC-U of Iowa-530HT) or 3.5 × 108 PFU/mL of recombinant Ad5CMVeGFP (Iowa Vector Core, VVC-U of Iowa-4). BMDCs were harvested 3 days later and stained with antibodies specific for CD11c (clone N418, BioLegend) and MHC-I (H-2Kb, clone AF6–88.5.5.3, eBioscience) and analysed by flow cytometry on a BD LSRII analyser (BD Biosciences). 104 CD11c+MHC-I+GFP+ BMDCs were then transplanted into the footpad of B6 hosts in 15 μls PBS.
Recipient animals were euthanized 7 days after BMDC transplant to collect spleen and the draining LN (popliteal). Organs were processed as described in5 (link) and samples were stained with antibodies specific for Thy1.1 (clone OX-7, BioLegend), Thy1.2 (clone 30-H12, BioLegend), CD4 (clone GK1.5, BioLegend) and CD8 (clone 53–6.7, BioLegend). Cells were then analysed on a BD LSRII flow cytometer (BD Biosciences).
Damo M., Fitzgerald B., Lu Y., Nader M., William I., Cheung J.F., Connolly K.A., Foster G.G., Akama-Garren E., Lee D.Y., Chang G.P., Gocheva V., Schmidt L.M., Boileve A., Wilson J.H., Cui C., Monroy I., Gokare P., Cabeceiras P., Jacks T, & Joshi N.S. (2020). Inducible de novo expression of neoantigens in tumor cells and mice with NINJA. Nature biotechnology, 39(1), 64-73.
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9

Analyzing Germinal Center Responses to Adjuvanted Vaccines

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Mice were s.c. immunized with alum-adsorbed NP–OVA (50 µg) and TLR7–alum or TLR7-NP (equivalent gardiquimod dose, 20 µg) in 100 µl PBS at tail base. Inguinal dLNs were excised at day 4, day 7, day 14 and day 22 to prepare single-cell suspensions. For flow cytometry analysis of GC B cells, follicular T cells (TFH) and plasmablasts, cells from the dLNs were stained with Ghost Dye Violet 510 (Tonbo Biosciences). Cells were then washed and blocked with Fc-blocker (clone 2.4G2, BD Bioscience) prior to staining with markers, including CD19 (clone 1D3/CD19, Biolegend), CD38 (clone 90, BD Biosciences), CD95 (clone Jo2, BD Biosciences), CD138 (clone 281-2, BD Biosciences), CD44 (clone IM7, BioLegend), CD3 (clone 17A2, BioLegend), CD4 (clone GK1.5, BioLegend), CXCR5 (clone L138D7, BioLegend) and PD1 (clone 29F.1A12, BioLegend). After staining, cells were washed and fixed with 1.5% PFA. Stained cells were collected using BD FACS Diva v8.01 software associated with a BD LSRII flow cytometer. Data were analysed with FlowJo 10 software. The gating strategy consisted of gating GCBC on live single CD3−CD19+CD95+CD38− cells, TFH on live single CD19−CD3+CD4+CXCR5+PD1hi, and plasmablasts on live single CD138+CD44+ cells.
Yin Q., Luo W., Mallajosyula V., Bo Y., Guo J., Xie J., Sun M., Verma R., Li C., Constantz C.M., Wagar L.E., Li J., Sola E., Gupta N., Wang C., Kask O., Chen X., Yuan X., Wu N.C., Rao J., Chien Y.H., Cheng J., Pulendran B, & Davis M.M. (2023). A TLR7-nanoparticle adjuvant promotes a broad immune response against heterologous strains of influenza and SARS-CoV-2. Nature Materials, 22(3), 380-390.
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10

Transgenic TCR Mice Thymus Analysis

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NINJA, NINJA-C and NINJA-F mice were crossed with either GP33-specific TCR Tg P14 mice or with GP66-specific TCR Tg SMARTA mice. Developing T cells were harvested from the thymus of 6–8 week old litters and analysed by flow cytometry on a BD Biosciences LSRII analyser after staining with antibodies specific for Thy1.2 (clone 30-H12, BioLegend), Thy1.1 (clone OX-7, BioLegend), CD8 (clone 53–6.7, BioLegend), CD4 (clone GK1.5, BioLegend).
Damo M., Fitzgerald B., Lu Y., Nader M., William I., Cheung J.F., Connolly K.A., Foster G.G., Akama-Garren E., Lee D.Y., Chang G.P., Gocheva V., Schmidt L.M., Boileve A., Wilson J.H., Cui C., Monroy I., Gokare P., Cabeceiras P., Jacks T, & Joshi N.S. (2020). Inducible de novo expression of neoantigens in tumor cells and mice with NINJA. Nature biotechnology, 39(1), 64-73.
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