Splenocytes or lung cells were prepared and counted from each mice in different groups as previously described, respectively [15 (link), 16 (link)]. Cells were plated in triplicate at 5 × 106 cells per well in a 24-well plate and incubated with CMFO (10 μg/mL) and anti-CD28/CD49d (1 μg/mL, eBioscience) overnight. RPMI 1640 medium used is a negative control. Cell responses were monitors through a cell stimulation cocktail (1 μg/mL, eBioscience). Cells were stained for anti-CD4-APC-Cy7 (Cat#552051, BD Biosciences), anti-CD8α-BV510 (Cat#563068, BD Biosciences), anti-CD44-FITC (Cat#561859, BD Biosciences), anti-CD62L-PerCP-Cy5.5 (Cat#560513, BD Biosciences), and intracellular markers anti-IFN-γ-PE (Cat#554412, BD Biosciences) and anti-IL-2-APC (Cat#554429, BD Biosciences). Absolute number of IFN-γ+ and IL-2+ T cells, central memory T cells (TCM), and effector memory T cells (TEM) was determined by an LSRII multicolor flow cytometer (BD Biosciences) and analyzed through FlowJo software. The results were shown as the mean ± SEM per group (n = 6).
Lsrii multicolor flow cytometer
Manufactured by BD
Sourced in United States
The BD LSRII multicolor flow cytometer is a high-performance analytical instrument designed for the detection and analysis of individual cells within a heterogeneous sample. The core function of the LSRII is to provide multidimensional data on the physical and fluorescent characteristics of cells, enabling researchers to identify and quantify specific cell populations of interest.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28 cd49d, by Thermo Fisher Scientific (3 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions)
Anti cd62l percp cy5, by BD (1 mentions)
Anti cd4 apc cy7, by BD (1 mentions)
Anti ifn γ pe, by BD (1 mentions)
Anti cd44 fitc, by BD (1 mentions)
Anti il 2 apc, by BD (1 mentions)
Anti il 2, by BD (1 mentions)
Gr1 fitc, by BD (1 mentions)
Cd4 pac blue, by BD (1 mentions)
Anti cd3 apc, by BD (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Anti b220 percp, by BD (1 mentions)
Cd11b apc, by BD (1 mentions)
Intracellular staining kit, by BD (1 mentions)
Cd3 pacblue, by BD (1 mentions)
Nk1.1 pe, by BD (1 mentions)
B220 percp, by BD (1 mentions)
Pi rnase staining buffer, by Abcam (1 mentions)
Anti ifn γ percp cy5, by Thermo Fisher Scientific (1 mentions)
6 protocols using lsrii multicolor flow cytometer
1
Phenotypic Profiling of Immune Cells
2
Multiparametric Analysis of Mouse Immune Cells
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Spleens were removed from mice and single-cell suspensions were prepared by mashing the spleen using a 3-ml syringe plunger on a strainer (70 μM) and washing cells with PBS. Single cell suspensions were stained for flow cytometric analysis with anti-CD3-APC, anti-CD4-Pac Blue, anti-CD8-Pac Orange, Anti- B220-Per CP, anti-annexin-FITC, and anti PI-PE (BD Pharmingen, San Diego, CA). Blood and peritoneal fluid were also harvested and single-cell suspensions were prepared. Cells were stained with Gr-1-FITC, B220-Per CP, CD11b-APC, CD3-Pac Blue, CD11c-PE-Cy7; or CD4-Pac Blue, CD8-Pac Orange, Ly49D-FITC, NK1.1-PE, CD127-APC.
To measure production of cytokines on a per cell basis, splenocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 30 ng/mL) and ionomycin (400 ng/mL) in the presence of 10 μg/mL of Brefeldin A. After 18 hours, cells were surface stained with anti-CD4 and anti-CD8 and processed with an intracellular staining kit (BD Biosciences) according to manufacturer’s instructions. Intracellular antibodies included anti-interferon (IFN)-γ (eBioscience, San Diego, CA), anti-TNF, and anti-IL-2 (both BD Biosciences). Data were acquired on a LSR II multicolor flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).
To measure production of cytokines on a per cell basis, splenocytes were stimulated with phorbol 12-myristate 13-acetate (PMA, 30 ng/mL) and ionomycin (400 ng/mL) in the presence of 10 μg/mL of Brefeldin A. After 18 hours, cells were surface stained with anti-CD4 and anti-CD8 and processed with an intracellular staining kit (BD Biosciences) according to manufacturer’s instructions. Intracellular antibodies included anti-interferon (IFN)-γ (eBioscience, San Diego, CA), anti-TNF, and anti-IL-2 (both BD Biosciences). Data were acquired on a LSR II multicolor flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).
3
Cell Cycle Analysis of C2C12 Myoblasts
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C2C12 myoblasts were plated at 2.5 × 105 cells/well in six-well plates in GM overnight, followed by induction of cell cycle arrest in G0/G1 phase by switching the medium to DMEM/high glucose with 1% FBS for 24 h. For cell cycle analysis, cells were then treated with GM, FGF9 (10 ng/mL) in GM. At 24 and 36 h. Cells were harvested with trypsin and fixed in 66% ethanol for cell cycle analysis. After fixing, cells were incubated with propidium iodide (PI)/RNase staining buffer (Abcam) for 30 min for cell cycle analysis using LSRII Multi-Color Flow Cytometer (BD Biosciences, San Jose, CA, USA), PI was excited with the 488 nm blue laser and imaged with the 575 ± 26 nm band–pass filter. Cell cycle population analyses were performed using FlowJo® software (Tree Star). Experiments included three biological replicates.
4
Intracellular Flow Cytometry for T Cell Analysis
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Intracellular flow cytometry analysis was performed as previously described (22 (link)). Briefly, 5 × 106 splenocytes were plated in each well of a 24-well plate and incubated with either CMFO (10 µg/mL) or PPD (10 µg/mL) in the presence of 1 µg/mL anti-CD28/CD49d (eBioscience CA, USA). RPMI1640 medium and cell stimulation cocktail (eBioscience, CA, USA) were used as negative and monitoring controls, respectively. Cells were stained with surface markers, including anti-CD4 PE Cy7, anti-CD8a PE, anti-CD44 APC-eFluor® 780, anti-CD62L FITC mAbs, and intracellular markers anti-IFN-γPerCP-Cy5.5 and anti-IL-2 APC mAbs (all from eBioscience, CA, USA). The stained cells were analyzed by an LSRII multicolor flow cytometer (BD Biosciences, CA, USA). The absolute number of CMFO-specific CD4+ or CD8+ IFN-γ positive TEM (effector memory T cells, CD62LloCD44hi) and CD4+ or CD8+ IL-2 positive TCM (central memory T cells, CD62LhiCD44hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA). The results are represented as mean ± SD per group (n = 6).
5
Multiparametric Flow Cytometry Analysis
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Cell suspensions were incubated with anti-mouse CD16/CD32 (Biolegend, 101,319). For cell surface flow cytometry, cells from spleens, thymuses and LNs were stained with specific antibodies for surface antigens as follows: PE-anti-CD4 (Biolegend, 100,408), Pacific Blue-anti-CD4 (Biolegend, 100,531), Brilliant Violet 510-anti-CD8a (Biolegend, 100,751), APC/Cy7-anti-TCRβ (Biolegend, 109,220), 7-AAD (BD Pharmingen™, 559,925), APC-anti-CD25 (Biolegend, 102,012), PerCP/Cy5.5-anti-CD44 (Biolegend, 103,032), PE/Cy7-anti-CD62L (Biolegend, 104,418), PE-anti-CD278 (ICOS) (Biolegend, 107,705), APC-anti-CD304 (Neuropilin-1, Nrp1) (Biolegend, 145,206), PE/Cy7-anti-CD279 (PD-1) (Biolegend, 109,110). For intracellular staining, cells were fixed and permeabilized with Fixation/Permeabilization Kit (eBioscience, 00–5123, 00–5223), washed with Permeabilization Buffer (eBioscience, 00–8333) and stained with PE-Cy7-anti-ki67 (eBioscience, 25-5698-82), PE-anti-CTLA4 (Biolegend, 106,306), AF488-anti-Foxp3 (Thermo Scientific™, 53-5773-82), PE-anti-Foxp3 (Biolegend, 126,404). For apoptosis analysis, cells were stained with FITC-AnnexinV (Biolegend, 640,906) in AnnexinV Binding Buffer (Biolegend, 422,201). Samples were analyzed by LSRII multicolor flow cytometer (BD Biosciences CA, USA) and data analysis was performed using the FlowJo software (Tree Star, USA).
6
Quantitative analysis of T cell response
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Spleen cells were further seeded in triplicates at 5 × 106 cells/well in a 24-well plate and stimulated with WH121 (10 μg/mL) in the presence of 1 μg/mL anti-CD28/CD49d (eBioscience CA, USA) for 20 h at 37°C under 5% CO2. For the last 4 h of the stimulation, 3 μg/mL brefeldin A and 2 μM monensin solution (eBioscience CA, USA) were added. The cells were washed in FACS buffer (1% FCS-PBS) and stained for 30 min at RT with anti-CD4-PE-Cy7 (clone GK1.5, eBioscience), anti-CD8α-PE (clone53-6.7, eBioscience), anti-CD62L-FITC (clone MEL-14, BD Pharmingen), and anti-CD44-APC-Cy7 (clone IM7, BD Pharmingen) mAbs. After the cells had been washed and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen CA, USA), the intracellular cytokines were stained with anti-IFN-PerCP-Cy5.5 (clone XMG1.2; eBioscience CA, USA) and anti-IL-2-allophycocyanin (clone JES6-5H4; eBioscience CA, USA) mAbs for 30 min at RT. The cells were washed twice, resuspended in FACS buffer and analyzed using an LSRII multicolor flow cytometer (BD Biosciences CA, USA). The absolute number of IFN-γand/or IL-2 positive CD4+ and CD8+ T cells, TCM (central memory T cells, CD62LhiCD44hi) and TEM (effector memory T cells, CD62LloCD44hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA) as previously described [25 (link)], respectively. The data are represented as the mean ± SD per group (n = 6).
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