BrdUFlowEx® FITC Kit

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应用范围
Flow Cytometry (FACS)
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原理 The BrdUFlowEx® Kit is designed for labeling and subsequent immunodetection of proliferating cells by flow cytometry. The kit can be used to analyze proliferation of human or mouse leukocytes and variety of human and murine cell lines.
品牌 BrdUFlowEx®
样品类型 Cell Samples
产品特性 BrdUFlowEx® FITC Kit unmasking method relies on oxidative attack at the deoxyribose moiety of DNA strand by copper (I) in the presence of oxygen. The unmasking principle so called "atomic scissors" was successfully used to detect BrdU by immunochemistry (Ligasova et al., 2012). The atomic scissors reaction causes mild DNA damage and allows simultaneous detection of other cellular nuclear proteins that are usually lost during other BrdU unmasking methods (e.g. PCNA-1). The reaction requires considerable oxygen supply which is achieved by shaking the reaction mixture. Following the DNA scission the cells are stained with fluorescently labeled BrdU specific antibody (Liboska et al., 2012). Test can be performed as bivariate analysis of BrdU incorporation and DNA content. 7-AAD is provided to stain the DNA. It allows the user to analyze BrdU incorporation and to asses of amount of cellular DNA according to the 7-AAD staining intensity (cells in G0/1, S and G2/M phase of the cell cycle). The method is compatible with the use of additional fluorescently labeled antibodies against other cell surface or intracellular molecules together with BrdU detection. The provided protocol was optimized for test tubes and 96-well Plates.
组件
  • Fix and Lysing Solution
    1 vial containing 10x concentrated fixation and erythrocyte lysing solution, the vial is intended for 100 reactions.
  • Permeabilizing Solution
    1 vial containing 30 ml of ready-to-use cellular membrane permeabilizing solution, the vial is intended for 100 reactions.
  • AS Solution 1
    (the abbreviation AS stands for atomic scissors), 4 vials containing lyophilized sodium ascorbate, 1 vial is intended for 25 reactions.
  • AS Solution 2
    (the abbreviation AS stands for atomic scissors), 1 vial containing 10 ml of ready-to-use copper (II) solution, the vial is intended for 100 reactions.
  • AS Solution 3
    (the abbreviation AS stands for atomic scissors), 2 vials containing 30 ml of ready-to-use buffer, one vial is intended for 50 reactions.
  • Anti-BrdU FITC
    1 vial containing 1 ml of ready-to-use solution containing FITC labeled anti-BrdU antibody (clone MoBu-1, IgG1), the vial is intended for 100 reactions.
  • 7-AAD
    1 vial containing 2 ml of ready-to-use 7-aminoactinomycin D solution, the vial is intended for 100 reactions.
  • BrdU
    4 vials containing lyophilized 5-bromo- 2-deoxyuridine, 1 vial is intended to prepare 2 ml of 10 mM BrdU solution.
试剂未包括
  • Deionized water (dH2O)
  • PBS (phosphate buffered saline)
  • Test tubes (12 x 75 mm) or 96-well plates
  • Rack for the test tubes
  • Centrifuge
  • Laboratory shaker
  • Automatic pipettes with disposable tips
  • Vortex
  • Flow cytometer - blue laser excitation 488 nm, light emission at 525 nm (FITC channel) and 675 nm (PerCP channel).
  • 50 mL polypropylene tubes or glass bottles for diluting and storing the 1x Fix and Lysing solution
背景 There are in principle four techniques how to assess cellular proliferation: (I) measurement the level of DNA synthesis, (II) fluorescence dilution assays, (III) detection of unique proliferation specific cellular markers, and (IV) measurement of cell biochemical activity. Among the mentioned methods the most reliable and accurate assay available today is to quantify the newly synthesized DNA by incorporation of labeled nucleosides or nucleoside analogues. BrdUFlowEx® FITC Kit labels cellular DNA in proliferating cells with the use of thymidine analogue 5-bromo-2- deoxyuridine (BrdU). The labeling can be performed in vitro by adding BrdU to culture medium or in vivo by exposing experimental animals to BrdU via intraperitoneal injections or drinking water. Long time exposures (several hours to overnight) are used for experiments with identification of cycling versus non-cycling populations, whereas short time exposures allow analysis of cell cycle kinetics (so called pulse-chase experiments). Once BrdU is incorporated within the double stranded DNA helix, it is masked by the pairing bases and inaccessible to anti-BrdU antibodies. There are several ways to unmask incorporated BrdU. Either the cellular DNA is exposed to denaturating agents or DNA cleaving enzymes are used to remove the masking bases. Stretches of single stranded DNA with exposed BrdU are incubated with fluorescently labeled antibody. Often the test is performed as bivariate analysis of BrdU detection and DNA content. Some protocols allow simultaneous detection of surface and intracellular molecules together with BrdU detection.
应用备注 Suggested Cell Number: 1 x107 cells/ ml
样本量 100 μL
试剂准备
  • Fix and Lysing Solution
    Dilute Fix and Lysing Solution ten times with deionized water, i.e. mix 1 part of the Fix and Lysing Solution with 9 parts of deionized water (e.g. 5 mL of Fix and Lysing Solution and 45 mL of dH2O). Unused diluted Fix and Lysing solution may be stored up to 4 weeks either at room temperature or at 2-8 °C.
  • AS Solution 1
    Reconstitute the content of the vial in 4 mL of PBS. Unused reconstituted AS Solution 1 may be frozen and stored up to 3 months at -20 °C or lower. The solution can be thawed and refrozen repeatedly.
  • BrdU
    Reconstitute the content of the vial in 2 mL of dH2O. The reconstituted BrdU stock solution is in 10 mM concentration (3.07 mg/ mL). Unused reconstituted BrdU solution may be frozen and stored up to 12 months at -20 °C or lower. The solution can be thawed and refrozen repeatedly.
样品收集 Harvesting the cells
  • Cells grown in dishes or flasks:
    • Bring the cells growing attached to plastic to suspension by trypsinization. If cells are grown in suspension proceed directly to the next step. Centrifuge the cells at 300 g for 10 min, discard supernatant.
    • Wash the cells by resuspending the pellet in 10 mL of PBS (room temperature), centrifuge at 300 g for 5-10 min and discard supernatant.
    • Resuspend the cells at ~ 1 x 107 cells /mL of PBS.
    • Dispense 0.1 mL of the cell suspension to test tubes.
  • Cells grown in 96-well plates (cells in suspension):
    • Centrifuge the plate with cells at 300 g for 5 minutes at room temperature, discard supernatant.
    • Add 200 μL PBS (room temperature), centrifuge the plate with cells at 300 g for 5 minutes, discard supernatant.
    • Resuspend the pellets in 20 μL PBS.
  • Whole blood cultures
    • Add 20 mM EDTA/PBS solution (not provided) to the whole blood cultures to final concentration 2 mM EDTA.
    • Incubate for 15 min at room temperature.
    • Mobilize the cells by vortexing/tapping the tube/flask and proceed to centrifugation step as with the cells grown in suspension (above).
样品制备

BrdU labeling:
Expose cells to BrdU by diluting the BrdU stock solution (10 mM) into the culture medium in final concentration ranging from 10-60 M. The exact protocol depends on the experiment design (pulse, long time labeling).

实验流程

Staining the cellular surface antigens

  • Add fluorescent antibodies to the test tubes/wells containing the harvested cells and mix.
  • Incubate for 15-30 minutes according to the specific requirements.
  • Wash the cells by adding 2 mL PBS to tubes / 200 ?l PBS to wells in plates.
  • Centrifuge at 300 g for 5 minutes and discard the supernatant.
BrdU detection:
Two following protocols are optimized either for reactions perfomed in test tubes or 96-well plates. The protocols differ in reagent volumes used in the procedure.

BrdU detection in test tubes
  1. Add 1 mL of the diluted Fix and Lysing Solution to the tubes containing 0,1 mL of cell suspension and mix (i.e. use the diluted Fix and Lysing Solution in volume that is equal to ten volumes of the resuspened cells).
  2. Incubate the mixture for 30 minutes at room temperature.
  3. Wash the cells by adding 2 mL of PBS, centrifuge the cells at 500 g for 5 minutes at room temperature and discard supernatant.
  4. Resuspend the cells in 0.3 mL of Permeabilizing Solution.
  5. Incubate for 15 minutes at room temperature.
  6. Wash the cells by adding 2 mL of PBS, centrifuge the tubes at 500 g for 5 minutes at room temperature and discard supernatant.
  7. Add 0.1 mL of PBS.
  8. Add 0.1 mL of AS Solution 1 and mix by gentle vortexing.
  9. Add 0.1 mL of AS Solution 2.
  10. Place the tubes on a shaker and incubate at 400 rpm for 10 minutes at room temperature.
  11. Add 0.4 mL of AS Solution 3.
  12. Incubate at 400 rpm for another 10 minutes at room temperature.
  13. Wash the cells by adding 2 mL of PBS, centrifuge the tubes at 500 g for 5 minutes at room temperature and discard the supernatant.
  14. Resuspend the pellet in 0.1 mL of AS Solution 3.
  15. Add 10 μL of Anti-BrdU FITC. Add other fluorescently labeled antibodies against intracellular antigens and vortex gently.
  16. Incubate for 30 minutes at room temperature protected from light.
  17. Wash the cells by adding 2 mL of PBS, centrifuge the tubes at 500 g for 5 minutes at room temperature and discard the supernatant.
  18. Resuspend the pellet in 0.2 mL of the diluted Fix and Lysing Solution.
  19. Add 20 μL of 7-AAD to stain cellular DNA (optional).
  20. Incubate for 20 minutes at room temperature in the dark.
  21. Measure with flow cytometer.


BrdU detection in 96-well plates
  1. Add 200 μL of the diluted Fix and Lysing Solution to well containing the resuspended cells (i.e. use the diluted Fix and Lysing Solution in volume that is equall to ten volumes of the resuspened cells).
  2. Incubate the mixture for 30 minutes at room temperature.
  3. Centrifuge the cells at 500 g for 5 minutes at room temperature. Discard supernatant. Wash by adding 200 μL of PBS. Centrifuge the cells at 500 g for 5 minutes at room temperature and discard supernatant.
  4. Add 50 μL of Permeabilizing Solution. Mix gently by tapping the plate several times.
  5. Incubate for 15 minutes at room temperature.
  6. Wash by adding 200 μL of PBS. Centrifuge the cells at 500 g for 5 minutes at room temperature and discard supernatant.
  7. Add 30 μL of PBS.
  8. Add 30 μL of AS Solution 1.
  9. Add 30 μL of AS Solution 2.
  10. Place the plate on a shaker and incubate at 400 rpm for 10 minutes at room temperature.
  11. Add 150 μL of AS Solution 3.
  12. Incubate at 400 rpm for 10 minutes at room temperature.
  13. Centrifuge the cells at 500 g for 5 minutes at room temperature. Discard the supernatant. Wash by adding 200 μL of PBS. Centrifuge the cells at 500 g for 5 minutes at room temperature and discard supernatant.
  14. Add 50 μL of AS Solution 3.
  15. Add 5 μL of Anti-BrdU FITC. Add other fluorescently labeled antibodies to intracellular antigens and mix the content by tapping the plate.
  16. Incubate at 300~400 rpm for 30 minutes at room temperature protected from light.
  17. Wash by adding 200 μL of PBS. Centrifuge the cells at 500 g for 5 minutes at room temperature and discard supernatant.
  18. Resuspend the pellet in 200 μL of the diluted Fix and Lysing Solution.
  19. Add 20 μL of 7-AAD to stain cellular DNA (optional).
  20. Incubate for 20 minutes at room temperature in the dark.
  21. Measure with flow cytometer.


Flow cytometric analysis:

Bivariate analysis of DNA and BrdU
  1. Adjust the PMT of SSC and FSC parameters so that the cell populations are on scale.
  2. Set the PerCP channel to linear scale and adjust the PMT so that cell populations are on scale.
  3. Adjust the PMT in FITC channel so that the negative populations fall into the lower third of the scale.
  4. Acquire 10 000 - 20 000 events per sample.
  5. Draw a gate around the target cell population.
  6. Display the gated cells in FSC-Height versus FSCArea and draw a diagonal gate that excludes doublets and aggregates.
  7. Display singlets in FITC vs PerCP dot plot and draw gates around the G0/G1, S and G2/M populations. The subG1 population sometimes seen in cellular cultures represents the apoptotic cells.
  8. Calculate the proportion of cells in each subpopulation.

限制 仅限研究用
注意事项
  • Wear protective gloves.
  • Do not use reagents after their expiration date.
  • Avoid contamination of reagents.
  • The flow cytometer should be calibrated on a routine basis using fluorescent microbeads to ensure stable sensitivity of detectors.
  • Any deviation from the recommended procedure can affect the results.
储存条件 4 °C