Lymph nodes were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were typically four μm thick and stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were boiled in citrate-based solution to retrieve antigens and subsequently quenched in 3% hydrogen peroxide. Antibodies used included anti-CD21 (Cat. ab75985, Abcam, Cambridge, MA, USA), MECA79 (Cat. 53-6036-80, eBioscience, San Diego, CA, USA), anti-PD-1 (Cat. AF1021, R&D systems, Minneapolis, MN, USA), anti-CD3 (Cat. ab5690, Abcam), anti-PAX5 (Cat. sc-1974, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD4 (Cat. 14–0042, eBioscience), anti-CD8 (Cat. 14–0081, eBioscience), anti-CD138 (Cat. AF3190, R&D Systems) and anti-HA (Cat. 3724, Cell Signaling Technology). Horseradish peroxidase-coupled secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were used in combination with DAB for visualization. For immunofluorescence staining, sections were stained with Alexa Fluor 488 conjugated anti-GL7 (Cat. 144611, BioLegend, San Diego, CA, USA) and anti-CD4 (Cat. 14–0042, eBioscience). Secondary antibody for anti-CD4 was goat anti-Rat IgG (H + L) Alexa Fluor 594 (Cat. A11007, Invitrogen, Carlsbad, CA, USA).
Anti cd4
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Austria
Anti-CD4 is a laboratory reagent used to detect and quantify CD4+ T cells, a type of lymphocyte that plays a crucial role in the immune system. The product is designed for research and clinical applications, and its core function is to provide a reliable and precise method for the identification and enumeration of CD4+ T cells.
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Lab products found in correlation
Anti cd8, by Thermo Fisher Scientific (52 mentions)
Anti cd3, by Thermo Fisher Scientific (35 mentions)
Anti cd45, by Thermo Fisher Scientific (28 mentions)
Anti cd11b, by Thermo Fisher Scientific (23 mentions)
Anti foxp3, by Thermo Fisher Scientific (21 mentions)
Anti cd11c, by Thermo Fisher Scientific (21 mentions)
Anti nk1, by Thermo Fisher Scientific (19 mentions)
Anti cd44, by Thermo Fisher Scientific (16 mentions)
Anti b220, by Thermo Fisher Scientific (15 mentions)
Anti f4 80, by Thermo Fisher Scientific (13 mentions)
Anti gr 1, by Thermo Fisher Scientific (13 mentions)
Anti cd8a, by Thermo Fisher Scientific (12 mentions)
Anti ifn γ, by Thermo Fisher Scientific (11 mentions)
Anti cd19, by Thermo Fisher Scientific (10 mentions)
Anti cd62l, by Thermo Fisher Scientific (10 mentions)
Anti ly6c, by Thermo Fisher Scientific (8 mentions)
Anti mhcii, by Thermo Fisher Scientific (8 mentions)
Anti cd86, by Thermo Fisher Scientific (8 mentions)
Anti cd3, by BioLegend (8 mentions)
Ionomycin, by Merck Group (8 mentions)
178 protocols using anti cd4
1
Multimodal Immunohistochemical Analysis of Lymph Nodes
2
Peripheral Blood T and M Cell Phenotyping
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For surface staining, peripheral blood samples were incubated with anti-CD4, anti-CD4, or F4/80 (eBioscience, San Diego, CA, USA) at 4 °C for 30 min. Then, cells were permeabilized for intracellular staining, which stained with anti-FOXP3, anti-IL-17A, anti-IFNγ, anti-IL-4, anti-inducible nitric oxide synthase (iNOS), and anti-CMAF (Santa Cruz) for Treg, Th17, Th1, Th2, M1 and M2 detections, respectively. Cells were acquired on Canto II flow cytometer (BD Biosciences), and data were analyzed with FlowJo v.9.5.2 software (Tree Star). Lastly, labeled cells were enumerated by Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed by BD FACSDiva™ Software 6.0 (BD).
3
Quantifying T-cell Infiltration in Tumors
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Tumors were harvested at an average tumor size of 150-200 mm3 and snap frozen. Tissues were cryosectioned at 8 µm and immunohistochemical (IHC) experiments were conducted as follows: slides were fixed in cold acetone or 4% formalin, blocked in 5% normal mouse serum (Jackson Immunoresearch) and incubated with primary antibodies for 1.5 h at room temperature. Primary antibodies were anti-CD3 (Dako, A0452, #280), anti-CD4 (Affymetrix, #14-0042) and anti-CD8a (Affymetrix, #14-0081). Sections were dried and stained with haematoxylin/eosin in a Leica ST4040 automatic stainer.
Assessment of T cell infiltration was performed in a blinded fashion and scored as 0 (negative), 1 (<150 cells per mm2), 1-2 (150-300 cells per mm2), 2 (300-500 cells per mm2, 2-3 (500-800 cells per mm2), and 3 (>800 cells per mm2). N=3 per syngeneic tumor model.
Assessment of T cell infiltration was performed in a blinded fashion and scored as 0 (negative), 1 (<150 cells per mm2), 1-2 (150-300 cells per mm2), 2 (300-500 cells per mm2, 2-3 (500-800 cells per mm2), and 3 (>800 cells per mm2). N=3 per syngeneic tumor model.
4
Analyzing T Cell Cytokine Production in Nlrc3 Knockout Mice
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Splenocytes from WT and Nlrc3−/− mice were treated with ACK lysis buffer at room temperature for 1 min to remove red blood cells. Splenocytes were washed, counted and plated at 2 × 105 cells per well in a 96-well plate coated with 1 µg/mL anti-CD3 (145-2C11, Affymetrix eBioscience) and 1 µg/mL anti-CD28 (16–0281, Affymetrix eBioscience). Cells were cultured at 37°C with and without 20 ng/ml murine IL-2 (212-12, Peprotech) for 4 days. Brefeldin A (00–4506, Affymetrix eBioscience) was added to the media for 3 h, followed by washing in PBS, and staining with anti-CD4 (14–0042–85, Affymetrix eBioscience) and anti-CD3 (145-2C11, Affymetrix eBioscience) antibodies on ice for 20 min. Stained cells were fixed in 1% paraformaldehyde for 30 min on ice and permeabilized using permeabilization buffer (00–8333-56, Affymetrix eBioscience) according to manufacturer’s instructions. To detect intracellular cytokines, fixed cells were stained with anti-IFN-γ (50–7311, Tonbo) and anti-TNF (506322, Biolegend) for 30 min on ice. Flow cytometry were performed as described above.
5
Analyzing T Cell Cytokine Production in Nlrc3 Knockout Mice
6
Immunophenotyping and Apoptosis Analysis
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Rat anti-mouse mAbs (Affymetrix eBioscience), which included fluorescein isothiocyanate- (FITC-) labeled anti-CD4 (0.3 μl, No11-0041) as well as phycoerythrin- (PE-) labeled anti-CD8a (0.7 μl, No11-0081), were utilized to stain cells from blood specimens for a 15 min period in dark. After adding hemolysin (250 μl), the cells were subject to further 15 min incubation in the dark and PBS rinsing thrice. CD4+/CD8+ ratio was determined as the ratio of average fluorescence intensity of CD4+ lymphocytes to that of CD8+ cells detected using the flow cytometer (Beckman coulter, Navios, USA).
THCA cells undergo certain treatments, including staining using Annexin V-FITC and propidium iodide (PI) Apoptosis Detection Kit I (BD, USA) in line with specific instructions, and cell apoptosis was analyzed through FACS (BD, USA).
Data analysis was completed using Cell Quest Research Software (BD, USA).
THCA cells undergo certain treatments, including staining using Annexin V-FITC and propidium iodide (PI) Apoptosis Detection Kit I (BD, USA) in line with specific instructions, and cell apoptosis was analyzed through FACS (BD, USA).
Data analysis was completed using Cell Quest Research Software (BD, USA).
7
Quantitative Immunohistochemical Analysis of Pancreatic Islets
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Consecutive 8 μm cryosections were prepared from 5 different levels of the pancreas and stained with optimal concentration of anti-CD4, anti-CD8 (both from Affymetrix; Santa Clara, Ca, USA), anti F4/80 and anti-FoxP3 (eBioscience, Nordic Biosite, Täby, Sweden). The slides were then incubated with polymer horseradish peroxidase-labeled secondary antibodies, and 3,3-Diaminobenzidine (DAB), respectively. The stained sections were analyzed in a Leica DMRX microscope. At least 40 islets were analyzed from each pancreas. The scoring of the extent of staining was performed as follows:
CD4/CD8: score 1, cells located peripherally, encircling the islet; score 2, cells infiltrating up to 1/3 of the islet; score 3, cells infiltrating 1/3 to 2/3 of the islet; score 4, cells infiltrating more than 2/3 of the islet.
F4/80: score 1, cells located peripherally, encircling the islet; score 2, discrete presence of cells in islet; score 3, moderate presence of cells in islet; score 4, marked presence of cells in islet.
FoxP3: score 1, only a few positive cells; score 2, low density of positive cells in islet; score 3, moderate density of cells in islet; score 4, high density of cells in islet.
CD4/CD8: score 1, cells located peripherally, encircling the islet; score 2, cells infiltrating up to 1/3 of the islet; score 3, cells infiltrating 1/3 to 2/3 of the islet; score 4, cells infiltrating more than 2/3 of the islet.
F4/80: score 1, cells located peripherally, encircling the islet; score 2, discrete presence of cells in islet; score 3, moderate presence of cells in islet; score 4, marked presence of cells in islet.
FoxP3: score 1, only a few positive cells; score 2, low density of positive cells in islet; score 3, moderate density of cells in islet; score 4, high density of cells in islet.
8
Mouse CRC Cell Line Culturing and Immunophenotyping
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The mouse CRC cell lines CT26 were obtained from the American Type Culture Collection (ATCC), and cultured in RPMI 1640 (Gibco) with 10% FBS (Australia origin) and 1% penicillin/streptomycin (Gibco) in a humidified atmosphere with 5% CO2 at 37 °C. The antibodies for FCM analysis including anti‐CD4, anti‐CD8, anti‐CD3, anti‐TCR beta, and MR1 were bought from Becton Dickinson (BD). The antibodies (anti‐CD4, anti‐CD8, anti‐rabbit IgG, anti‐mouse IgG) for indirect immunofluorescence (IF) analysis were bought from Thermo Fisher Scientific (Waltham).
9
Cytokine Profile of Allogeneic T Cells
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In total, 1 × 106 lymphocytes derived from the spleens and lymph nodes of MHC-matched C57BL/6 mice were cocultured with a total of 1 × 105 DCs or DCTP for three days. Subsequently, the T lymphocytes were collected and stained with rat anti-mouse monoclonal antibodies including anti-CD3e (Thermo, Waltham, MA), anti-CD4 (Thermo, Waltham, MA), anti-CD8a (Thermo, Waltham, MA), anti-IL4 (Cyto-Fast™ Fix/Perm Buffer Set, Biolegend, San Diego, CA), anti-IFN-γ (Cyto-Fast™ Fix/Perm Buffer Set, Biolegend, San Diego, CA), and anti-FOXP3 (True-Nuclear™ Transcription Factor Buffer Set, Biolegend, San Diego, CA), followed by FCM analysis. Moreover, cells stained with the anti-IL4 and anti-IFN-γ antibodies were pretreated with a Cell Activation Cocktail kit following the kit instructions (Biolegend, San Diego, CA). The culture supernatant was harvested, and the IFN-γ (Thermo, Waltham, MA), IL-2 (Thermo, Waltham, MA), IL-10 (Thermo, Waltham, MA), and TGF-β (Thermo, Waltham, MA) levels were measured by ELISAs.
10
Quantification of Peripheral Blood Cells
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For quantification of peripheral blood cells, 50 µl of whole blood was collected. Red blood cells were lysated and Fc- blockage was performed (Fc-Block, Pharmingen). After washing cells were stained with anti-B220 (BD Pharmingen), anti-CD8a (BioLegend), anti-CD11b (BD-Pharmingen), anti-CD4, anti-CD115, anti-Nk1.1, and anti-Ly6c (all ThermoFisher Scientific). To collect absolute numbers, CountBright Absolute Counting Beads (ThermoFisher Scientific) were added. Samples were measured on a FACSCanto II (BD Bioscience, Franklin Lakes, USA) and analyzed with FlowJo (Tree Star, Ashland, USA).
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