Tumors were harvested and chopped into several pieces. The tumor pieces were digested into single cell suspensions by incubating in digestion buffer [2 mg/mL collagenase D (Merck) and 40 μg/mL DNase I (Merck)] for 1 h at 37 °C. Cell suspensions were filtered using a 70-μm cell strainer (Corning) and a 40-μm nylon mesh to remove cell clumps. After washing with FACS buffer (1% FBS in PBS), cells were primed with antibodies targeting CD45 (30-F11, BD Pharmingen), CD4 (RM4-5, BD Pharmingen), CD8 (53-6.7, BD Pharmingen), CD3 (17A2, eBioscience), or ICOS (7E.17G9, eBioscience). Next, the cells were permeabilized using a permeabilization kit (eBioscience) and stained for IFN-γ (eBioscience). Data was acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using the FlowJo software (Tree Star Inc., Ashland, OR, USA).
Rm4 5
Manufactured by BD
Sourced in United States
The RM4-5 is a laboratory centrifuge designed for general-purpose applications. It features a maximum speed of 4,500 rpm and a maximum relative centrifugal force of 3,024 x g. The centrifuge accommodates a variety of sample volumes and tube sizes. For detailed specifications, please consult the product documentation.
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Lab products found in correlation
Fjk 16, by Thermo Fisher Scientific (11 mentions)
Facsdiva software, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Tc11 18h10, by BD (3 mentions)
Facscanto 2 system, by BD (3 mentions)
Ebio17b7, by Thermo Fisher Scientific (3 mentions)
Cd8 53 6, by BD (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Foxp3 egfp b6 mice, by Jackson ImmunoResearch (2 mentions)
Rpmi 1640 with 10 fbs and antibiotics, by Thermo Fisher Scientific (2 mentions)
Mouse ccl22, by R&D Systems (2 mentions)
Facsaria 3 sorter, by BD (2 mentions)
Cd44 im7, by BioLegend (2 mentions)
Facsdiva, by BD (2 mentions)
Mel 14, by BD (2 mentions)
Ionomycin, by BD (2 mentions)
Ifn γ xmg1.2, by BioLegend (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Golgistop, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
42 protocols using rm4 5
1
Tumor Infiltrating Immune Cell Profiling
2
MSCV-based Retroviral Vector Targeting CD4
3
Murine CD4+ T Cell Dynamic Imaging
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Murine splenic CD4+ T cells were first stained with CFSE, according to the manufacturer’s recommendations (Invitrogen) and then with rat anti-mouse CD4-APC (RM4-5, BD Biosciences) mAb. After staining, cells were incubated in ibidi’s μ-Dish (ibidi, Nanterre, France) in the thermo-regulated chamber of the Zeiss Axiovert 100M confocal microscope. A time-lapse acquisition was performed with a delay of 120 s between frames. Each frame was illuminated using 488-nm and 633-nm lasers, and fluorescence was collected using 505- to 580-nm and 644- to 719-nm filters. The magnification was 40×/1.3 oil, and images were saved as 8 bits. The image stack was splitted using ImageJ, v1.43, and images were analyzed using CellProfiler 2.0.10415. The cell segmentation was performed on the CFSE fluorescence, and the fluorescence of the CD4 staining was analyzed using R software. Intensity was normalized to the maximum fluorescence measured at t0 for each condition.
4
Treg Migration and Suppression Assay
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Splenocytes (1 × 106 cells/well) from FoxP3-eGFP/B6 mice (The Jackson Laboratory, Bar Harbor, ME) were plated in 5 Nm pore size transwells (Cell Biolab, Inc., San Diego, CA, USA). RPMI 1640 with 10% FBS and antibiotics (Life Technologies, Grand Island, NY, USA) was added to the outer well in the presence or absence of 100-500ng/ml mouse Ccl22 (R&D Systems, Minneapolis, MN, USA). Cells were allowed to migrate for 2 hrs before harvesting cells from the bottom well (Klarquist et al, 2010 (link); Justus et al, 2014 ). Cells from top and bottom wells and a splenocyte sample not included in the migration assay were subjected to immunofluorescent staining to detect Treg, using 145-2C11, RM4-5, and PC61 antibodies to CD3, CD4, and CD25, respectively (BD Biosciences, San Jose, CA, USA). Splenocytes were sorted using a BD FACS Aria III sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Sorted Treg from reporter mice were combined with effector cells from TCR transgenic mice recognizing gp10022-35 (Pmel1 mice) or human tyrosinase368-376 (h3TA2 mice) in IFN-gamma ELISPOT plates in the presence of cognate peptide. The resulting suppression by Treg cells was quantified by comparing to the spot numbers produced by the same number of effector cells without Treg cells.
5
Treg Quantification in B16-BL6 Tumors
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To assess the Treg population by flow cytometry, the B16-BL6 tumors were harvested at 5 days following the final Tregs injection. Dissociated tumors were prepared as previously described [30 (link)]. Cells were stained as previously described [34 (link)]. Briefly, the cells were stained with peridinin chlorophyll protein-CY5.5-conjugated anti-CD4 Ab (cat. no. 550954, clone RM4-5, BD Biosciences), phycoerythrin-conjugated anti-CD25 Ab (cat. no. 12-0251-81, clone PC61.5, eBioscience, San Diego, CA, USA), and allophycocyanin-conjugated anti-Foxp3 Ab (cat. no. 17-5773-82, clone FJK-16s, eBioscience). Samples were analysed using a BD Biosciences BD FACScanto II flow cytometry analyser and FACSDiva software (BD Biosciences).
6
Treg Migration and Suppression Assay
7
Immunohistochemical Analysis of Retinal Cells
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Anesthetized mice were perfused with 20 mL of PBS. Eyes were enucleated and fixed in 4% paraformaldehyde in 2× PBS for 15 min and then transferred to 2× PBS on ice for 10 min. After dissecting eyes, retinal whole mounts were prepared. Retinas were then transferred to ice-cold methanol and kept at −80 °C until use.
For immunohistochemistry, retinas were first blocked in a blocking buffer (0.3% Triton, 0.2% BSA, and 5% goat serum in PBS) for 1 h at room temperature and incubated with primary antibodies and Alexa Fluor 647-conjugated isolectin GS-B4 (1:100; Thermo Fisher Scientific) overnight at 4 °C. After washing, retinas were incubated with secondary antibodies for 4 h at 4 °C. Retinas were mounted after washing. Rabbit anti-P2ry12 antibody (1:500; a gift from H. Weiner, Brigham and Women’s Hospital), rat anti-CD11b antibody (1:100, clone M1/70; Abcam), rat anti-MHC class II (1:1,000, I-A/I-E; BD Pharmingen), rat anti-CD4 (1:200, RM4-5; BD Pharmingen), and rat anti-CD8a (1:200, 53-6.7; BD Pharmingen) were used for primary antibodies. Alexa Fluor 594-conjugated goat anti-rabbit antibody, and Alexa Fluor 488-conjugated goat anti-rat antibody (1:500; Thermo Fisher Scientific) were used for secondary antibodies.
For immunohistochemistry, retinas were first blocked in a blocking buffer (0.3% Triton, 0.2% BSA, and 5% goat serum in PBS) for 1 h at room temperature and incubated with primary antibodies and Alexa Fluor 647-conjugated isolectin GS-B4 (1:100; Thermo Fisher Scientific) overnight at 4 °C. After washing, retinas were incubated with secondary antibodies for 4 h at 4 °C. Retinas were mounted after washing. Rabbit anti-P2ry12 antibody (1:500; a gift from H. Weiner, Brigham and Women’s Hospital), rat anti-CD11b antibody (1:100, clone M1/70; Abcam), rat anti-MHC class II (1:1,000, I-A/I-E; BD Pharmingen), rat anti-CD4 (1:200, RM4-5; BD Pharmingen), and rat anti-CD8a (1:200, 53-6.7; BD Pharmingen) were used for primary antibodies. Alexa Fluor 594-conjugated goat anti-rabbit antibody, and Alexa Fluor 488-conjugated goat anti-rat antibody (1:500; Thermo Fisher Scientific) were used for secondary antibodies.
8
Cytokine Profiling of Activated Splenocytes
9
Comprehensive Immune Cell Profiling
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Cells from the thymus, spleen or lymph nodes were resuspended in staining buffer at a density of 2 × 106 cells/ml. The cells were incubated with anti-FcγRII/III monoclonal antibody (mAb) (2.4G2), followed by antibodies against CD4 (RM4-5, BD Pharmingen), CD8 (53–6.7, BD Pharmingen), B220 (RA3-6B2, BioLegend), Thy1.1 (53–2.1, BioLegend), Thy1.2 (OX-7, BioLegend), CD44 (IM7, BioLegend), CD62L (MEL-14, BioLegend), Dll1 (HMD1-5, eBiosciences), Dll4 (HMD4-1, eBiosciences), Jagged1 (HMJ1-29, eBiosciences) or Jagged2 (HMJ2-1, eBiosciences). After gating out cells that were positive for 7-AAD, excluding the doublets, the fluorescence intensity of 105 cells was measured with a FACS Canto II flow cytometer (BD Biosciences, CA) and analyzed with FACSDiva (BD Biosciences) or FlowJo (Tree Star) software programs. For intracellular staining, cells were fixed with an intracellular staining kit (72–5775, eBioscience), permeabilized and stained with Foxp3 (3G3, Tonbo Biosciences) antibody.
10
Immunophenotyping and Cell Sorting Analysis
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