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IngoFlowEx® Kit

ABIN3072914 产品详细信息, 供应商: Log in to see
应用范围
Flow Cytometry (FACS)
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原理 IngoFlowEx® Kit is designed for quantification of phagocytic activity of human granulocytes and monocytes by measuring the ingestion of fluorescently labeled E. coli bacteria in human heparinized whole blood using flow cytometry.
品牌 IngoFlowEx®
样品类型 Blood Cells
组件
  • E. coli-FITC (Ready-to-Use)
    One vial containing 1 ml of fluorescein-labeled human plasma opsonized E. coli strain K-12 in suspension, the vial is intended for 100 reactions.
  • Quenching Solution (Ready-to-Use)
    One vial containing 20 ml of buffered solution of trypan blue, dark blue solution, the vial is intended for 200 reactions.
  • 10x Lysing Solution (10x concentrated solution)
    One vial containing 60 ml of 10x concentrated transparent solution of fixation-lysing reagent. Each vial is intended to prepare 600 ml of 1x Lysing Solution, i.e. 200 reactions.
  • 25x Wash Buffer (25x concentrated solution)
    One vial containing 80 ml of concentrated wash buffer. Each vial is intended to prepare 2 liters of 1x Wash buffer, i.e. 222 reactions.
  • DNA Staining Solution (Ready-to-Use)
    One amber vial containing 60 ml of buffered propidium iodide solution. Each vial is intended for 200 reactions.
试剂未包括
  • Deionized water (dH2O), approximately 0.6 liter for one kit
  • Phosphate buffered saline (PBS), approximately 2 liters
  • Suitable 5 mL test tubes for blood staining (e.g. 12 x 75 mm)
  • Racks for the test tubes
  • Centrifuge with suitable rotor for 5 mL tubes
  • Automatic pipettes with disposable tips
  • Vortex
  • Thermal incubator or water bath (37 °C)
  • Container with ice (ice cubes or crushed ice) suitable for placing a rack with tubes
  • Flow cytometer - blue laser excitation 488 nm, light emission at 525 nm (FITC channel) and 617 nm (PE channel).
  • Cylinders and glass bottles for the dilution of 25x Wash Buffer and of 10x Lysing Solution
背景 Phagocytosis is a part of the innate defense mechanisms in which phagocytes ingest extracellular particulate material. The ability of phagocytosis is a characteristic feature of the so-called professional phagocytic cells (neutrophil and eosinophil granulocytes, monocytes and macrophages). The process includes particle binding, its ingestion and subsequent degradation. Plasma proteins (C3b and antibodies) assist the phagocytes in particle recognition and ingestion by covering the surface of foreign particles with readily recognizable ligands.

The test is based on measuring the fluorescence of cells that ingested FITC-labeled E. coli. A sample of heparinized blood is mixed with fluorescent E. coli and incubated at 37 °C. A control reaction without E. coli is performed in parallel with each reaction with E. coli. This negative control is used to set the discrimination boundery between the phagocyting and the nonphagocyting cells. The non-ingested bacteria are separated by repeated washes. The fluorescence of remaining extracellular and surface bound bacteria is quenched with trypan blue, a vital dye does not cross the cellular membrane. Samples are then subjected to erythrocyte lysis and fixed. Finally the cellular DNA is stained with propidium iodide. DNA staining helps to define nucleated cells from debris and clumps of bacteria. Bacteria used in the test were opsonised with human AB plasma, the results are not primarily dependent on opsonizing activity of the tested blood sample.
应用备注
  • Blood must be collected into a tube containing heparin. Anticoagulants EDTA and citrate negatively affects results of the analysis.
  • Blood samples should be measured within 8 hours after collection.
  • The flow cytometer should be calibrated on a routine basis using fluorescent microbeads to ensure the stable sensitivity of detectors.
  • Do not use reagents after the expiration date stated on vial labels.
  • Avoid contamination of reagents.
  • Use protective gloves and follow procedures for handling potentially infectious materials.
  • Avoid contact of samples with skin, eyes and mucous membranes.
样本量 50 μL
试剂准备

Preparation of reagents before measurement

  • Wash Buffer
    Dilute 25x Wash Buffer with PBS before use. Allow the buffer to cool to 2-8 °C. Assign with the date of preparation and store at 2-8 °C for up to 4 weeks.
  • Lysing Solution
    Dilute 10x Lysing Solution with deionized water. Assign with the date of preparation and store at 2-8 °C or at room temperature for up to 4 weeks.
  • E. coli-FITC
    Before use thoroughly mix the content of the vial using vortex mixer.

样品制备

Pipetting protocol

  • Allow 1x Wash Buffer to cool to 2-8 °C.
  • Allow 1x Lysing Solution to reach room temperature.
  • Place E. coli-FITC, Quenching Solution and DNA Staining Solution on ice.
  • Place a rack for test tubes on ice.

For each blood sample prepare two tubes:
  • the reaction with E. coli
  • the control reaction (without E. coli)
  1. Pipette 50 μL of blood into the test tubes.
  2. Place the tubes on ice for 10 minutes.
  3. Add 10 μL of E. coli-FITC to the tube intended to perfom reaction with Ecoli and mix using a vortex mixer. Return the tube to ice.
  4. Do not add anything into the control tubes.
  5. Transfer the tubes (including the control tubes) into a thermal incubator set to 37 °C (or a water bath set to 37 °C).
  6. Incubate for 30 minutes (10 minutes if using the water bath).
  7. Return the tubes to ice and incubate for 5 minutes.
  8. Add 100 μL of Quenching Solution (2-8 °C) and mix well. Keep the tubes on ice.
  9. Add 3 mL of 1x Wash Buffer (2-8 °C) and centrifuge the tubes at 200 g for 5 min.
  10. Remove the supernatant by aspiration into a container with an appropriate disinfectant. Do not disturb the pellet.
  11. Repeat wash (steps 9 and 10).
  12. Resuspend the pellet in the residual volume of 1x Wash buffer.
  13. Add 2 mL of 1x Lysing Solution (room temperature), mix and incubate for 20 minutes at room temperature in the dark.
  14. Centrifuge the cells at 200 g for 5 min.
  15. Remove the supernatant but do not disturb the pellet.
  16. Add 3 mL precooled 1x Wash buffer and centrifuge at 200 g for 5 min.
  17. Remove supernatant and resuspend the pellet in 300 μL of DNA Staining Solution (2-8 °C).
  18. Incubate for 10 minutes on ice in the dark.
  19. Measure with flow cytometer within 2 hours after adding the DNA Staining Solution.

实验流程

Setting up the cytometer before the first run: (Optional)

The color compensation for spectral overlap may be assessed with performing a set of single reagent stained blood samples. To perform single stained reactions follow the assay procedure described in section 8. Use only one kit component at a time.

  1. blood and Ecoli-FITC (omit Quenching Solution and DNA Staining Solution)
  2. blood and Quenching Solution (omit E. coli-FITC and DNA Staining Solution)
  3. blood and DNA Staining Solution (omit E. coli-FITC and Quenching Solution).
Use these measurements to adjust the cytometer settings.

Analysis of samples
Set a gate in PE channel and acquire a sufficient number of cellular PE-bright events (at least 10,000 events). This gate excludes debris and clumps of bacteria from further analysis. Visualize the PE channel gated events in the side-scatter (SSC) versus forward-scatter (FSC) dot-plot and set gates around the granulocytes and monocytes. Then bring the gated subpopulations to histograms in FITC channel. Use the control reaction histogram to place the discrimination line. A single discrimination line setting is usually applicable for all tested samples within the run. Calculate the percentage of positive cells (this parameter is elswere called phagocytic activity).

限制 仅限研究用
储存条件 4 °C