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Lymphocyte m

Manufactured by Cedarlane
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The Lymphocyte M is a laboratory equipment designed for the analysis and enumeration of lymphocytes, a type of white blood cell. It provides accurate and reliable data on lymphocyte counts, which are crucial for the assessment of various medical conditions and immune system function. The core function of the Lymphocyte M is to perform this specialized cell analysis, facilitating clinical decision-making and patient care.

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Lab products found in correlation

Facscalibur flow cytometer, by BD (3 mentions) Gr 1 rb6 8c5, by BD (2 mentions) Cd11b m1 70, by Thermo Fisher Scientific (2 mentions) Cd44 im7, by Thermo Fisher Scientific (2 mentions) Cd62l mel 14, by Thermo Fisher Scientific (2 mentions) Epcam g8, by Thermo Fisher Scientific (2 mentions) Facscalibur machine, by BD (1 mentions) Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions) Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Collagenase, by Thermo Fisher Scientific (1 mentions) Amphotericin b, by Merck Group (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Hyaluronidase, by Merck Group (1 mentions) Hrp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions) Calf thymus dna, by Merck Group (1 mentions) Goat anti mouse igg, by Southern Biotech (1 mentions) Poly l lysine (pll), by Merck Group (1 mentions) Cd25 pc61, by BD (1 mentions) Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions) Cd3 145 2c11, by BioLegend (1 mentions)

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Lymphocyte-M gradient (2 citations)

7 protocols using lymphocyte m

1

Spleen and Lymph Node Single-Cell Analysis

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Spleen and lymph node samples were forced through 100 µm filters to prepare single-cell suspensions. Dead cells were removed by gradient centrifugation with lymphocyte M (Cedarlane, Burlington, NC, USA) and live cells were stained with surface antibodies for flow cytometric analysis. Data were collected in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Antibodies used: Gr-1 (RB6-8C5, BD Biosciences, San Jose, CA, USA), CD11b (M1/70, eBioscience, San Diego, CA, USA), CD62L (MEL-14, eBioscience, San Diego, CA, USA), CD44 (IM-7, eBioscience, San Diego, CA, USA) and EpCAM (G8.8, eBioscience, San Diego, CA, USA).
Tang W., Wang H., Ha H.L., Tassi I., Bhardwaj R., Claudio E, & Siebenlist U. (2016). The B-cell tumor promoter Bcl-3 suppresses inflammation-associated colon tumorigenesis in epithelial cells. Oncogene, 35(48), 6203-6211.
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2

Isolation and Characterization of hGBM Cells

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The hGBM cells were placed in ice-cold HBSS solution after being taken from the patient's brain. The specimens were, on average, larger than 50 mm3. Lymphocyte-M (Cedarlane labs) was used to remove the red blood cells from the specimen. A solution of 2% papain and dispase was used to gently dissociate the intact hGBM cells, followed by gentle grinding (trituration). FACSCalibur machine (BD Biosciences) was then used to sort out the cells. Clonal expansion and formation of orthotopic tumors was observed in both CD133+ and CD133− fractions. Cells from the CD133+ fraction were then used in the experiments.
Mahmood M.A., Hasan M.R., Khan U.J., Allen P.B., Kim Y.T., Ellington A.D, & Iqbal S.M. (2015). One-step tumor detection from dynamic morphology tracking on aptamer-grafted surfaces. Technology, 3(4), 194-200.
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3

Cytotoxicity Assay for OT-I and Tumor-specific CTLs

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To analyze OT-I cell cytotoxicity, EG7 or EG7-B7H4 cells (2 × 104) were labeled with 3 μM CFSE as target cells, and then incubated with activated OT-I cells for 24 h at various effector-to-target ratios. To obtain tumor-specific cytotoxic T lymphocytes (CTLs), dendritic cells and CD8 T cells were isolated from the spleens and draining lymph nodes of GL261-bearing mice on day 7, respectively, using negative isolation microbeads (Miltenyi Biotec). CD8 T cells cocultured with tumor lysate pulsed-dendritic cells for 3 days. Viable CD8 T cells were purified with Lymphocyte-M (Cedarlane) and incubated with CFSE-labeled target cells (GL261/GL261-B7H4) for 24 h. Killing effect was evaluated by a cell death marker (LIVE/DEAD® Fixable Dead Cell Stain kits, Thermo Scientific, USA) using flow cytometry. To observe the killing effect of CTLs under microscope, target cells (GL261/GL261-B7H4) were stained with 5 μM acetoxymethyl esters (AM, Thermo Scientific, USA) and coculture with tumor-specific T cells for 24 h. Live cell imaging and data analysis were performed using a Zeiss LSM 880 laser-scanning confocal microscope.
Zhou L., Ruan M., Liu Y., Zhu Y., Fu D., Wu K, & Zhang Q. (2019). B7H4 expression in tumor cells impairs CD8 T cell responses and tumor immunity. Cancer Immunology, Immunotherapy, 69(2), 163-174.
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4

Spleen and Lymph Node Single-Cell Analysis

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Spleen and lymph node samples were forced through 100 µm filters to prepare single-cell suspensions. Dead cells were removed by gradient centrifugation with lymphocyte M (Cedarlane, Burlington, NC, USA) and live cells were stained with surface antibodies for flow cytometric analysis. Data were collected in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Antibodies used: Gr-1 (RB6-8C5, BD Biosciences, San Jose, CA, USA), CD11b (M1/70, eBioscience, San Diego, CA, USA), CD62L (MEL-14, eBioscience, San Diego, CA, USA), CD44 (IM-7, eBioscience, San Diego, CA, USA) and EpCAM (G8.8, eBioscience, San Diego, CA, USA).
Tang W., Wang H., Ha H.L., Tassi I., Bhardwaj R., Claudio E, & Siebenlist U. (2016). The B-cell tumor promoter Bcl-3 suppresses inflammation-associated colon tumorigenesis in epithelial cells. Oncogene, 35(48), 6203-6211.
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5

Isolation and Culture of Primary Colorectal Cancer Cells

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Collection and processing of tissue specimens were performed with approval from the institutional review board of SMG-SNU Boramae Medical Center (IRB No.: 26-2015-42). Briefly, primary CRC specimens were obtained from patients undergoing surgical resection at SMG-SNU Boramae Medical Center (Seoul, Korea). Immediately after resection, the tumor specimens were placed in transport medium consisting of McCoy’s 5A medium supplemented with 3% penicillin/streptomycin, and 1.5 μg/mL amphotericin B (Sigma Aldrich, St. Louis, MO, USA). Within 2 h of removal, excess fat and normal tissues were removed from each specimen, and tissue fragments were washed with PBS and then finely minced. Enzymatic digestion was performed with McCoy’s 5A-based medium supplemented with 1.5 mg/mL collagenase (Thermo Fisher Scientific, Waltham, MA, USA), and 20 μg/mL hyaluronidase (Sigma Aldrich) for 1–2 h at 37 °C in a shaking incubator. To isolate single cells from the tissue fragments, the suspension was filtered through a 40-μm-pore size nylon cell strainer and washed with McCoy’s 5A medium. To remove erythrocytes and cell debris, lymphocyte centrifugation (Lymphocyte-M; Cedarlane, ON, Canada) was performed. The cells were then washed with PBS and cultured in McCoy’s 5A medium supplemented with 10% FBS, 6% penicillin/streptomycin, and 3 μg/mL amphotericin B.
Lee J., Kim Y.S., Lee J., Heo S.C., Lee K.L., Choi S.W, & Kim Y. (2016). Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness. Nutrients, 8(7), 439.
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6

Immunocyte ELISA Assay Protocol

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Similarly, we pre-coated 96-well multi-screen plates with 1 μg/ml goat anti-mouse IgG (Southern Biotech) or 20 μg/ml poly-L-lysine (Sigma) together with 20 μg/ml calf thymus DNA (Sigma) (Millipore, Billerica, USA) and then used 2% FCS in PBS to block the plates [20] . Single-cell suspensions of lymphocyte M (Cedarlane Laboratories Ltd., Hornby, Canada)-purified PBMCs at 0.25×10 5 cells/well, bone marrow cells (2 femurs) at 1×10 5 cells/well and splenic cells at 0.5×10 5 cells/well were added to the plates and incubated for 1 hour at 37 °C. The plates were washed, and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) was added to the plates and incubated at 20 °C for 1 hour.
Liu S., Sun L., Wang C., Cui Y., Ling Y., Li T., Lin F., Fu W., Ding M., Zhang S., Lei C, & Hu S. (2019). Treatment of murine lupus with TIGIT-Ig. Clinical immunology (Orlando, Fla.), 203.
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7

Isolation and Analysis of Lymphocytes

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Spleen and lymph node samples were forced through 100 μm filters to prepare single-cell suspensions. Dead cells were removed by gradient centrifugation with lymphocyte M (Cedarlane, Burlington, NC, USA) and live cells were stained with surface antibodies for FACS analysis. To isolate lymphocytes from colon, less than 0.1 cm colon pieces were cut and digested at 37°C with 3 mg mL−1 dispase II (Thermo Fisher Scientific, Waltham, MA, USA), 1 mg mL−1 collagenase D (Roche) and 0.1 mg mL−1 DNase I (Roche) for 1 h until no visible pieces were present. The digested tissue was passed through a 100 μm filter, followed by centrifugation on a Percoll gradient.
For intracellular staining, the cells were first stained with antibodies for surface marker expression, then permeabilized and stained with antibodies for intracellular protein for 1 h using Foxp3 Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA). Data were collected in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (BD, Franklin Lakes, NJ, USA). Antibodies to the following markers were used: CD4 (RM4–5, Thermo Fisher Scientific), CD3 (145–2C11, Biolegend, Dedham, MA, USA), TCRβ (H57–597, BD Biosciences), RORγt (AFKJS-9, Thermo Fisher Scientific), CD25 (PC61, BD Biosciences), Foxp3(FJK-16s, Thermo Fisher Scientific).
Tang W., Saret S., Tian R., Wang H., Claudio E., Murphy P.M, & Siebenlist U. (2021). Bcl-3 suppresses differentiation of RORγt+ regulatory T cells. Immunology and cell biology, 99(6), 586-595.
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