RBMRNA-405-induced effects on proliferation and dynamics of immune cell populations were evaluated using Accuri C6 (BD Biosciences). For T-cell subset analysis, 1×106 mice splenocytes (100 μl) were seeded per well into 96-well plates. Following two washes with PBS, the viability of cells was determined using Zombie Green™ Fixable Viability Kits (BioLegend, 423111). After washing once with FC buffer (PBS + 2% FBS), cells were resuspended in FC buffer. Then, splenocytes were stained with PerCP/Cyanine5.5 anti-mouse CD3 (BioLegend, 100218), PE anti-mouse CD8a (BioLegend, 100708), APC anti-mouse CD4 (BioLegend, 100412), PE anti-mouse CD62L (BioLegend, 161203), and APC anti-mouse/human CD44 (BioLegend, 103011). For GC response analysis, inguinal and iliac lymph nodes (LNs) were pooled and lymphocytes were isolated. Cells were treated with the same method as splenocytes except for the following antibodies: APC anti-mouse CD4 (BioLegend, 100412), PE anti-mouse CD185 (CXCR5) (BioLegend, 145503), PerCP/Cyanine5.5 anti-mouse CD278 (ICOS) (BioLegend, 117424), APC anti-mouse CD19 (BioLegend, 115512), PE anti-mouse CD95 (Fas) (BioLegend, 152608), PE anti-mouse CD138 (Syndecan-1) (BioLegend, 142504), APC anti-mouse/human CD45R/B220 (BioLegend, 103212), and PE anti-mouse CD79b (Igβ) (BioLegend, 132804).
Apc anti mouse cd4
Manufactured by BioLegend
Sourced in United States
The APC anti-mouse CD4 is a fluorochrome-conjugated antibody that binds to the CD4 surface receptor on mouse T helper cells. It is used for the identification and enumeration of CD4+ T cells in flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti mouse cd3, by BioLegend (5 mentions)
Accuri c6, by BD (3 mentions)
Apc anti mouse cd8a, by BioLegend (3 mentions)
Zombie green fixable viability kit, by BioLegend (2 mentions)
Pe anti mouse cd62l, by BioLegend (2 mentions)
Percp cyanine5.5 anti mouse cd3, by BioLegend (2 mentions)
Apc anti mouse human cd44, by BioLegend (2 mentions)
Apc anti mouse cd19, by BioLegend (2 mentions)
Cell staining buffer, by BioLegend (2 mentions)
Percp anti mouse cd8, by BioLegend (2 mentions)
Pe anti mouse ifn γ, by BioLegend (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Pe anti mouse cd8a, by BioLegend (1 mentions)
Apc anti mouse human cd45r b220, by BioLegend (1 mentions)
Pe anti mouse cd138 syndecan 1, by BioLegend (1 mentions)
Pe anti cd62l, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Moflo high performance cell sorter, by Beckman Coulter (1 mentions)
22 protocols using apc anti mouse cd4
1
Immune Cell Dynamics in RBMRNA-405 Treatment
2
Splenic Cell Sorting for Naive and CD45RBhi T Cells
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Splenic cells were prepared and stained with relevant surface antibodies and incubated for 30 min at 4°C. For naive T cell sorting, cells were stained with anti-mouse CD4-APC (Biolegend), anti-CD25-Percp 5.5 (Biolegend), anti-CD62L-PE ( Biolegend), and DAPI (Invitrogen). For CD45RBhi T cell sorting, cells were stained with anti-mouse CD4-APC (Biolegend), anti-CD45RB-PE (Biolegend), and DAPI. After incubation, cells were washed and suspended in 2% FBS in PBS. Cells were then sorted by BD FACSAriaTM Fusion Cell Sorter.
3
Quantifying T Cell Populations in Wound Healing
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In order to analyse the CD4+ and CD8 + T cell content in mouse spleen and lymph nodes during wound healing, the spleen and lymph nodes of the sacrificed mice on day 7 were removed in a sterile environment, washed with PBS, and then ground on a 0.75-μm sieve. The obtained cell suspension was washed with PBS, centrifuged at 1000 rpm for 5 min and then lysed with red blood cell lysis buffer (Solarbio). The cells were stained with cell staining buffer (Biolegend), incubated with antibodies of APC anti-mouse CD4 (BioLegend), brilliant violet 510TM anti-mouse CD8 (BioLegend), FITC anti-mouse CD3 (BioLegend) and finally analysed by flow cytometry (MoFlo High-Performance Cell Sorter, Beckman).
4
Detailed Multicolor Flow Cytometry Protocol
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APC/Cyanine7 anti-mouse Ly-6G/Ly-6C (Gr-1) clone RB6-8C5, Brilliant Violet 421™ anti-mouse/human CD11b clone M1/70, Brilliant Violet 785™ anti-mouse/human CD45R/B220 clone RA3-6B2, PE/Cy7 anti-mouse CD8a clone 53-6.7 and APC anti-mouse CD4 (BioLegend). The visualization of the flow cytometry data was performed with the “pseudocolor” or “contour” modes of Flowjo analysis software v10, as shown in Supp. Figs. 1 , 3 -5 . Gating of the hematopoietic subsets was performed using internal negative control cell populations, as defined by the “Guidelines for the use of flow cytometry and cell sorting in immunological studies” [59 (link), 60 (link)], and was applied uniformly to all samples analyzed.
5
Cell Death and Immune Cell Analysis
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HEK293T cells stained Alexa Fluor 647-conjugated annexin V, 1:50 (BioLegend) and propidium iodide (1 µg/mL) in DMEM (Nacalai Tesque, 08459-64) with 10 mM HEPES on room temperature for 15 min. Samples were filtered through a 37 µm mesh prior to cytometry analysis.
Splenocytes were collected from adult and P9, P5 mice, and P5 interspecies chimeras by mashing the spleen between glass slides and chilling it in PBS(–). Splenocytes were stained with APC anti-mouse CD45 (1:50, Biolegend 100516), PE anti-mouse CD3ε (1:50, Biolegend 100308), Alexa Fluor 488 anti-mouse CD3ε (1:50, Biolegend 100321), APC anti-mouse CD4 (1:50, Biolegend 100516), PerCP anti-mouse CD8 (1:50, Biolegend 100732), APC anti-rat CD45 (1:50, Biolegend 202212), PE anti-rat CD3ε (1:50, Biolegend 201412), APC anti-rat CD4 (1:50, Invitrogen 17-0040-82), and PerCP anti-rat CD8 (1:50, Biolegend 201712) in 50 µL of 0.1% BSA/PBS at 4 °C for 30 min. The samples were resuspended in 500 µL of 0.1% BSA/PBS and filtered through a 37 µm mesh prior to flow cytometry analysis.
Flow cytometry was conducted using a BD Accuri C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Becton Dickinson).
Splenocytes were collected from adult and P9, P5 mice, and P5 interspecies chimeras by mashing the spleen between glass slides and chilling it in PBS(–). Splenocytes were stained with APC anti-mouse CD45 (1:50, Biolegend 100516), PE anti-mouse CD3ε (1:50, Biolegend 100308), Alexa Fluor 488 anti-mouse CD3ε (1:50, Biolegend 100321), APC anti-mouse CD4 (1:50, Biolegend 100516), PerCP anti-mouse CD8 (1:50, Biolegend 100732), APC anti-rat CD45 (1:50, Biolegend 202212), PE anti-rat CD3ε (1:50, Biolegend 201412), APC anti-rat CD4 (1:50, Invitrogen 17-0040-82), and PerCP anti-rat CD8 (1:50, Biolegend 201712) in 50 µL of 0.1% BSA/PBS at 4 °C for 30 min. The samples were resuspended in 500 µL of 0.1% BSA/PBS and filtered through a 37 µm mesh prior to flow cytometry analysis.
Flow cytometry was conducted using a BD Accuri C6 flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Becton Dickinson).
6
Flow Cytometry Analysis of Antigen-Specific T Cells
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Flow cytometry analysis was performed using a BD Accuri 6 plus (BD Biosciences) and analyzed by FlowJo software (Tree Star, Ashland, OR, USA). Epitope- specific T cells were studied using MHC Class I Pentamers (F093-84C-E, ProImmune, Oxford, UK). Other antibodies used included the following: murine Fc block CD16/32 (101320, Biolegend); FITC anti-mouse CD8 (A5402-3bE, ProImmune); PE/Cy7 anti-mouse CD19 (115520, Biolegend), FITC anti-mouse CD11c (117306, Biolegend), APC anti-mouse H-2Kb bound to SIINFEKL(116619, Biolegend), PE anti-mouse CD370 (143504, Biolegend), PE anti-mouse PD-1 (135206, Biolegend), PE anti-mouse CD68 (137013, Biolegend), APC anti-mouse CD4 (100412, Biolegend). All staining procedures were performed according to the manufacturer’s recommendations. Gating strategies are shown in supplementary Fig. 12 .
7
Identification of Breast Cancer Stem Cells
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For detecting only BCSCs in cancers, cancer cells were stained with specific marker proteins using antibodies such as FITC anti-CD44 and APC anti-CD24 (BD, San Jose, CA, USA) to define CD44+/CD24-BCSCs. Single-cell suspensions were prepared from mouse tumor tissues or TDLNs to analyze the T-cells. The processes of tissue sample preparation are described in “Subcutaneous tumor and TDLN resection and sample preparation ” section. Cells were stained with specific antibodies as APC anti-mouse CD8a, APC anti-mouse CD4, and FITC anti-mouse CD3 (BioLegend, San Diego, CA, USA) to define CD8+/CD3 + cytotoxic T-cells or CD4+/CD3 + helper T-cells. The samples were analyzed by flow cytometry (Accuri C6, BD Biosciences, East Rutherford, NJ, USA).
8
Tumor Immune Profiling by Flow Cytometry
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Mice were sacrificed and tumors were harvested. 0.3 mg tumor tissue was isolated and added into a solution of collagenase (400 units per mL), DNase 1 (100 μg mL−1), and hyaluronidase (0.04 units per mL). The mixture was shaked under 37 °C for 1 h. After, the mixture was passed through a 70 μm nylon cell strainer. The sample was washed 3 times with PBS (with 2% FBS) and then resuspended in 1 × 106 cells per mL for flow cytometry analysis. Antibody (AF488-anti mouse CD45, PE/Cy7 anti-mouse CD3, APC anti-mouse CD4, BV510-anti mouse CD8, PE anti-mouse FOXP3 and PE anti-mouse IFN-γ, all purchased from BioLegend) was added following the antibody manufacturer's recommendations and incubated for 30 min on ice. Cells were analyzed on BD FACS Celesta, and data were analyzed using FlowJo 10. Cellular events were first gated by forward and then by side scatter characteristics. The cell counts for the CD45+, CD45+CD3+, CD45+CD3+CD4+, CD45+CD3+CD4+FOXP3+ CD45+CD3+CD8+ and CD45+CD3+CD8+IFN-γ+ T lymphocytes were assessed. For quantification, the proportion of each immune subpopulation was determined by flow cytometry. This number was then multiplied by the total number of cells in the tumor, which had been determined before flow cytometry by counting with a hemocytometer. To determine cell density, cell counts were normalized to tumor weight (cell number per gram tumor).
9
Tumor Cell Immunophenotyping by Flow Cytometry
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Tumor cells from LLC model were resuspended to a density of 1 × 106 cells/100 μL. The cells were first blocked with anti-mouse CD16/32 for 20 min at 4 °C and then stained with PE anti-mouse CD3 (100204, BioLegend, San Diego, CA, USA), APC anti-mouse CD4 (100312, BioLegend), APC anti-mouse CD8a (100712, BioLegend), APC anti-mouse CD19 (152410, BioLegend), APC anti-mouse NK1.1 (108710, BioLegend), FITC anti-mouse CD25 (101908, BioLegend), or PE anti-mouse FOXp3 (126404, BioLegend) purchased from BioLegend (USA). After incubation of 20 min, cells were washed and terminated with 1 mL PBS, and the stained cells were detected by flow cytometry (Accuri™ C6 CSampler, BD Biosciences, San Jose, CA, USA).
10
Splenic T Cell Phenotyping in IMQ-Induced Mice
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The proportion of Th1 and Th17 cells among the splenic CD4+ T cells of IMQ-induced mice was determined by flow cytometry. In brief, single-cell suspensions were obtained using filter grinding and fluorescent-activated cell sorting lysing solution. Then, the cells were stimulated for 5 h with phorbol myristate acetate (Sigma, St. Louis, MO, USA), ionomycin (Sigma), and protein transport inhibitor (Sigma). Then, the cells were incubated with FITC anti-mouse CD3 (Biolegend, San Diego, CA, USA) and APC anti-mouse CD4 (Biolegend) for 30 min on ice in the dark. After fixation with 2% formaldehyde for 20 min and permeabilization for 30 min, the cells were stained with PE anti-mouse IL-17A (Biolegend) and PerCp anti-mouse IFN-γ (Biolegend) on ice for an additional 30 min in the dark. Cells were analyzed using ACEA NovoCyte (ACEA Biosciences, San Diego, CA, USA) and data were analyzed using the FlowJo software (Tree Star, San Carlos, CA, USA).
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