Welcome guest, Sign in

Home

X
加载中

Endosomes embraces different set of compartments such as early endosomes, intermediate endosomes and late endosomes or lysosomes. They become acidic as they mature. This acidification is generated by the vacuolar membrane proton pump V-ATPase that is recruited in late endosomes. This protocol described the measurement of endosomal pH using dextran molecules labelled with pH sensitive and insensitive dyes.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Endosomal pH Measurement in Bone Marrow Derived Dendritic Cells

Immunology > Immune cell function > Dendritic cell
Authors: Sophia Maschalidi
Sophia MaschalidiAffiliation 1: INSERM, Unité 1013, Paris, France
Affiliation 2: Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France
Bio-protocol author page: a685
 and Bénédicte Manoury
Bénédicte ManouryAffiliation 1: INSERM, Unité 1013, Paris, France
Affiliation 2: Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France
For correspondence: benedicte.manoury@inserm.fr
Bio-protocol author page: a346
Vol 3, Iss 14, 7/20/2013, 2306 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.819

[Abstract] Endosomes embraces different set of compartments such as early endosomes, intermediate endosomes and late endosomes or lysosomes. They become acidic as they mature. This acidification is generated by the vacuolar membrane proton pump V-ATPase that is recruited in late endosomes. This protocol described the measurement of endosomal pH using dextran molecules labelled with pH sensitive and insensitive dyes.

Materials and Reagents

  1. CO2 independent medium (Invitrogen, catalog number: 18045054)
  2. Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich, catalog number: I3390)
  3. 10% fetal bovine serum (FBS) (Hyclone/PAA, catalog number: sv143-03)
  4. Penicillin-streptomycin (100 Units/ml, 100 μg/ml) (Sigma-Aldrich, catalog number: P11-010)
  5. Glutamine (Sigma-Aldrich, catalog number: G75013)
  6. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250)
  7. Granulocyte-macrophage colony stimulating factor (GM-CSF) (Peprotech, catalog number: 315-03)
  8. 40,000 MW Dextran fluorescein (10 mg/ml) (Molecular Probes)
  9. 40,000 MW Dextran Alexa 647 (10 mg/ml) (Molecular Probes)
  10. 5 mM EDTA (Invitrogen, catalog number: 15575-038)
  11. 1% Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153)
  12. Triton X-100 (Sigma-Aldrich, X-100) kept at room temperature
  13. 1x PBS (see Recipes)
  14. Conditioned complete medium (see Recipes)

Equipment

  1. Water bath (37 °C)
  2. Incubator (37 °C)
  3. The FACSCalibur flow cytometer (Becton Dickinson)
  4. Hemocytometer
  5. Centrifuge

Procedure

  1. Detach bone marrow-derived dendritic cells (BMDCs) with 1x PBS-5 mM EDTA (10 min at 37 °C).
  2. Wash the cells with 1x PBS twice by centrifugation at 367 x g for 10 min.
  3. Count cells. A total of 3 x 106 cells are required for pH measurement at different time points (kinetics of 10 min, 20 min, 30 min and 60 min for example). Additionally, 6 x 106 cells are needed to acquire the pH standard curve.
  4. For measurement of pH at different time points, resuspend the cells in 100 μl total volume of prewarmed conditioned complete medium containing 1 mg/ml of fluorescein- and 0.5 mg/ml Alexa-647-labeled 40,000 MW dextrans. Pulse cells in a water bath set at 37 °C for 10 min.
  5. Stop the reaction by adding a large volume (1 ml) of cold 1x PBS-1% BSA and wash cells extensively (6 times) with the same buffer to get rid of the non-internalised dextrans.
  6. After washing, resuspend the cells in prewarmed conditioned complete medium (1 ml) and incubate at 37 °C for different time points (chase). The pulse (10 min) corresponds to early endosomes (EE), the chase of 40 min corresponds to intermediate endosomes (IE) and the 110 min of chase corresponds to the lysosomes.
  7. Stop the reaction each time by immediately adding cold PBS and placing the tubes on ice.
  8. Rapidly, analyse the cells by FACS, via a FL1/FL4 gate selective for cells that have endocytosed both fluorescent probes and determine the ratio of the mean fluorescence intensity (MFI) emission between the two probes.
  9. For the pH standard curve, resuspend the cells in 200 μl total volume of prewarmed conditioned complete medium containing 1 mg/ml of fluorescein- and 0.5 mg/ml Alexa-647-labeled 40,000 MW dextrans and pulse the cells in a water bath set at 37 °C for 20 min. Repeat step 5 and split cells into nine 1.5 ml Eppendorf tubes for a pH measurement ranging from 4 to 8.
  10. Prepare several buffers that differ in pH by 0.5 units using prewarmed CO2 independent medium. Adjust the pH with citric acid or NaOH.
  11. Resuspend each cell pellet in a different pH solution supplemented with 0.001% of Triton X-100 to slightly permeabilise the cells and to give access to the external prefixed pH solution into the cell.
  12. Analyse immediately by FACS and determine the ratio of the mean fluorescence intensity (MFI) emission between the two fluorescent probes at each pH. Make the standard curve by plotting the different MFI ratio values that correspond to each pH and apply this formula to the MFI ratio values obtained before (steps 1-8, Figure 1).





    Figure 1. Kinetic of endo-lysosomal pH in BMDCs pulsed with a mixed of fluorescein- and Alexa-647-labeled 40,000 MW dextrans for 10 min and then chased for different times

Recipes

  1. 1x PBS
    137 mM NaCl
    2.7 mM KCl
    8 mM Na2HPO4
    1.46 mM KH2PO4
    Keep 1x PBS cold
  2. Conditioned complete medium
    IMDM supplemented with
    10% FBS
    100 Units/ml,100 μg/ml penicillin-streptomycin
    2 mM glutamine
    50 μM 2-mercaptoethanol
    10 ng/ml GM-CSF

Acknowledgments

This protocol is adapted from Savina et al. (2010); Maschalidi et al. (2012) and Sepulveda et al. (2009).

References

  1. Maschalidi, S., Hassler, S., Blanc, F., Sepulveda, F. E., Tohme, M., Chignard, M., van Endert, P., Si-Tahar, M., Descamps, D. and Manoury, B. (2012). Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation. PLoS Pathog 8(8): e1002841.
  2. Savina, A., Vargas, P., Guermonprez, P., Lennon, A. M. and Amigorena, S. (2010). Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays. Methods Mol Biol 595: 383-402.
  3. Sepulveda, F. E., Maschalidi, S., Colisson, R., Heslop, L., Ghirelli, C., Sakka, E., Lennon-Dumenil, A. M., Amigorena, S., Cabanie, L. and Manoury, B. (2009). Critical role for asparagine endopeptidase in endocytic Toll-like receptor signaling in dendritic cells. Immunity 31(5): 737-748.


How to cite this protocol: Maschalidi, S. and Manoury, B. (2013). Endosomal pH Measurement in Bone Marrow Derived Dendritic Cells. Bio-protocol 3(14): e819. DOI: 10.21769/BioProtoc.819; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook