For flow cytometry analysis cells were stained with CD14-PE-Cy7 (1:20, BD Bioscience, Breda, the Netherland), TLR4-APC (1:10, ebioscience, San Diego, USA) or left untreated. Cell numbers were quantified using the BD FACS Cantoll flowcytometer, histograms were generated using flowjo software version 7.6.2 (Treestar Inc, Ashland-OR, USA.
Facs cantoll flow cytometer
Manufactured by BD
Sourced in United States, China
The BD FACS Canto II is a flow cytometer that can be used to analyze and sort cells. It is capable of detecting and measuring multiple parameters of individual cells within a sample. The instrument uses lasers, optics, and electronics to provide quantitative analysis of the physical and fluorescent characteristics of cells.
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Lab products found in correlation
8 protocols using facs cantoll flow cytometer
1
Flow Cytometry Analysis of CD14 and TLR4
2
MUC1* Cell Surface Expression Analysis
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Example 2
Recognition of MUC1* on the cell surface was analyzed by surface staining of the cells using FACS. For MIN-C2 antibody 50 μl of a 10 μg/ml solution of purified antibody was bound individually to MUC1-negative HCT116 cells transfected with empty vector (HCT116-VEC8), or transfected with MUC1* expressing vector (HCT-MUC1*-10) and MUC1 positive ZR-75-1 cells at 4 degrees Celsius for 30 minutes. Cells were washed twice, and treated with 10 μg/ml anti-mouse-PE at 4 degrees Celsius for 30 minutes. Cells were washed twice, fixed in 2% formaldehyde in PBS, and analyzed using a BD FACS Cantoll flow cytometer (
3
Cell Surface Expression of MUC1*
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Example 2
Recognition of MUC1* on the cell surface was analyzed by surface staining of the cells using FACS. For MIN-C2 antibody 50 μl of a 10 μg/ml solution of purified antibody was bound individually to MUC1-negative HCT116 cells transfected with empty vector (HCT116-VEC8), or transfected with MUC1* expressing vector (HCT-MUC1*-10) and MUC1 positive ZR-75-1 cells at 4 degrees Celsius for 30 minutes. Cells were washed twice, and treated with 10 μg/ml anti-mouse-PE at 4 degrees Celsius for 30 minutes. Cells were washed twice, fixed in 2% formaldehyde in PBS, and analyzed using a BD FACS Cantoll flow cytometer (
4
Evaluating Lymphocyte Subsets in Clinical Cohorts
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Subjects had standard complete blood counts (CBCs) performed at the NIH Clinical Center in the Department of Laboratory Medicine. Lymphocyte (T cell, B cell, NK cell) flow cytometry quantification was performed using the BD FACS Cantoll flow cytometer. The following parameters were collected on most patients, but were removed in downstream analysis for the given reasons:
Three samples were removed due to inconsistencies found in their data (the sum of the absolute counts of cells from the TBNK assay was highly inconsistent with the total lymphocytes from the complete blood counts).
Absolute counts of leukocytes (including TBNK) were used for downstream analysis. The neutrophil to lymphocyte ratio (NLR), the ratio between the neutrophil absolute counts and lymphocyte absolute counts, was included as an additional CBC parameter for classification due to its previously described association with multiple medical conditions such as infections and cancer4 ,5 .
Three samples were removed due to inconsistencies found in their data (the sum of the absolute counts of cells from the TBNK assay was highly inconsistent with the total lymphocytes from the complete blood counts).
Absolute counts of leukocytes (including TBNK) were used for downstream analysis. The neutrophil to lymphocyte ratio (NLR), the ratio between the neutrophil absolute counts and lymphocyte absolute counts, was included as an additional CBC parameter for classification due to its previously described association with multiple medical conditions such as infections and cancer4 ,5 .
5
Immunophenotyping of Tumor-Infiltrating Cells
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BALF cells and intra-tumor lymphocytes were isolated from tumor-bearing mice treated with GalCer. Cell viability and number were assessed by trypan blue exclusion. For flow cytometry, 2 × 105 cells were stained using a standard protocol. The following antibodies (Abs) were used: APC-labeled anti-mouse CD4 mAb (clone GK1.5; eBiosciences, San Diego, CA), PE-Cy7-labeled anti-mouse CD8 mAb (clone 53-6.7; eBiosciences, San Diego, CA), PE-Cy7-labeled anti-mouse CD11b mAb (clone M1/70; eBiosciences), and FITC-labeled anti-mouse Ly-6G mAb (clone RB6-8C5; BD Biosciences). Samples were acquired using a FACSCanto ll flow cytometer and data analysis was performed using FACSDiva software (BD Biosciences).
6
Cell Cycle Analysis of Cancer Cells
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The cell cycle progression of cancer cell lines was evaluated using a PI cell cycle Kit. Cells were harvested by trypsin digestion, washed twice in PBS, and fixed in 75% precooled ethanol for more than 2 h. Fixed cells were then treated with kit reagents according to the manufacturer’s protocol and analyzed using FACSCantoll flow cytometer (BD FACSCanto II, China).
7
Grass Pea Genome Size Estimation
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Grass pea genome size was estimated following the procedure described by Dolezel et al.80 (link). Fresh, young leaf tissue (40 mg) of grass pea (LS007) and P. sativum (semi-leafless, obtained from a local market in Nairobi) was sliced finely using a scalpel blade while immersed in 2 mL of ice-cold Galbraith buffer (45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 0.1% w/v Triton X-100, pH 7). Three biological replicates were prepared for each grass pea and pea. Supernatants were filtered through one layer of Miracloth (pore size 22–25 µm). One aliquot of 600 µL was prepared from each replicate, along with three grass pea + pea mixes at 2:1, 1:1 and 1:2 ratios, respectively. Propidium iodide was added to each tube to a concentration of 50 µM. Reactions were incubated for 1 h on ice before measuring on a FACSCantoll flow cytometer (Becton Dickinson) with flow rates adjusted to 20–50 events/s. Results were analysed using FCSalyser (v. 0.9.18 alpha), using the gating strategy shown in Supplementary Fig. 10 . Grass pea genome size was estimated from the mixed sample by dividing the mean of the PE-A peak corresponding to grass pea nuclei by the mean of the PE-A peak corresponding to pea nuclei and multiplying by 4.45 Gbp, the estimated genome size of pea53 (link).
8
Grass Pea Genome Size Estimation
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Grass pea genome size was estimated following the procedure described by Dolezel et al. (2007) (link).
Fresh, young leaf tissue (40 mg) of grass pea (LS007) and Pisum sativum (semi-leafless, obtained from a local market in Nairobi) was sliced finely using a scalpel blade while immersed in 2 mL of icecold Galbraith buffer (45 mM MgCl 2 , 30 mM sodium citrate, 20 mM MOPS, 0.1% w/v Triton X-100, pH 7). Three biological replicates were prepared for each grass pea and pea. Supernatants were filtered through one layer of Miracloth (pore size 22-25 µm). One aliquot of 600 µL was prepared from each replicate, along with three grass pea + pea mixes at 2:1, 1:1 and 1:2 ratios, respectively.
Propidium iodide was added to each tube to a concentration of 50 µM. Reactions were incubated for 1 h on ice before measuring on a FACSCantoll flow cytometer (Becton Dickinson) with flow rates adjusted to 20-50 events/s. Results were analysed using FCSalyser (v. 0.9.18 alpha). Grass pea genome size was estimated from the mixed sample by dividing the mean of the PE-A peak corresponding to grass pea nuclei by the mean of the PE-A peak corresponding to pea nuclei and multiplying by 4.300 Gbp, the estimated genome size of pea (Leitch et al. 2019) .
Fresh, young leaf tissue (40 mg) of grass pea (LS007) and Pisum sativum (semi-leafless, obtained from a local market in Nairobi) was sliced finely using a scalpel blade while immersed in 2 mL of icecold Galbraith buffer (45 mM MgCl 2 , 30 mM sodium citrate, 20 mM MOPS, 0.1% w/v Triton X-100, pH 7). Three biological replicates were prepared for each grass pea and pea. Supernatants were filtered through one layer of Miracloth (pore size 22-25 µm). One aliquot of 600 µL was prepared from each replicate, along with three grass pea + pea mixes at 2:1, 1:1 and 1:2 ratios, respectively.
Propidium iodide was added to each tube to a concentration of 50 µM. Reactions were incubated for 1 h on ice before measuring on a FACSCantoll flow cytometer (Becton Dickinson) with flow rates adjusted to 20-50 events/s. Results were analysed using FCSalyser (v. 0.9.18 alpha). Grass pea genome size was estimated from the mixed sample by dividing the mean of the PE-A peak corresponding to grass pea nuclei by the mean of the PE-A peak corresponding to pea nuclei and multiplying by 4.300 Gbp, the estimated genome size of pea (Leitch et al. 2019) .
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