Shreffler:Milk Program

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(Procedure)
(Procedure)
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'''Basophil Assay and Isolation of Mononuclear Cells''' (done simultaneously)
'''Basophil Assay and Isolation of Mononuclear Cells''' (done simultaneously)
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##Label 4 (50mL) tubes as PBS: Blood, AIM-V, PBMC, Ficoll and 2(15mL) tubes as Basophil blood and RPMI
#Bring 15/10mL of Ficoll(sterile) to room temperature and put 5mL RPMI in water bath.   
#Bring 15/10mL of Ficoll(sterile) to room temperature and put 5mL RPMI in water bath.   
#Collect pre-made stimulants from the freezer (Rm 40 in clear boxes with rubber bands). LABEL each one
#Collect pre-made stimulants from the freezer (Rm 40 in clear boxes with rubber bands). LABEL each one
#Collect 6 epindorph tubes.  Label 1 of these tubes "basophil cocktail." Make a cocktail as follows: Combine 180 uL RPMI + 3.6 uL IL-3.  MIX WELL.
#Collect 6 epindorph tubes.  Label 1 of these tubes "basophil cocktail." Make a cocktail as follows: Combine 180 uL RPMI + 3.6 uL IL-3.  MIX WELL.
#Label the 5 remaining tubes "Basophil medium."  Add 30 uL of the cocktail into these tubes.
#Label the 5 remaining tubes "Basophil medium."  Add 30 uL of the cocktail into these tubes.
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#Label 4 (50mL) tubesThese tubes can be found in styrafoam container above the benchLabel as follows:
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#Add 270 uL of the warmed RPMI to the five basophil tubes(made above), fMLP, milk extract, and anti-IgE.
 +
#Add 135 uL of RPMI to PMA and CaI and then combine them.
 +
# Prepare a 10-fold dilution of milk stimulants:
 +
    aTransfer 270 uL of basophile medium from the aliquotes prepared in step 2 to each of the milk 2-5 epindorph tubes.
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    bTransfer 30 uL from tube "milk 1" to "milk 2".  VORTEX.
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    c.  Take 30 uL from "milk 2" and add to "milk 3."  ETC.  VORTEX after each step.
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a. PBS: Blood
 
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b. AIM-V
 
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c. PBMC
 
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d. Ficoll
 
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#
 
#
#
#
#

Revision as of 17:20, 27 July 2009

Contents

Overview

CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.


T regulatory Assay

Materials

  • 15 mL polypropylene tubes
  • PBS
  • 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.

Procedure

Basophil Assay and Isolation of Mononuclear Cells (done simultaneously)

    1. Label 4 (50mL) tubes as PBS: Blood, AIM-V, PBMC, Ficoll and 2(15mL) tubes as Basophil blood and RPMI
  1. Bring 15/10mL of Ficoll(sterile) to room temperature and put 5mL RPMI in water bath.
  2. Collect pre-made stimulants from the freezer (Rm 40 in clear boxes with rubber bands). LABEL each one
  3. Collect 6 epindorph tubes. Label 1 of these tubes "basophil cocktail." Make a cocktail as follows: Combine 180 uL RPMI + 3.6 uL IL-3. MIX WELL.
  4. Label the 5 remaining tubes "Basophil medium." Add 30 uL of the cocktail into these tubes.
  5. Add 270 uL of the warmed RPMI to the five basophil tubes(made above), fMLP, milk extract, and anti-IgE.
  6. Add 135 uL of RPMI to PMA and CaI and then combine them.
  7. Prepare a 10-fold dilution of milk stimulants:
   a.  Transfer 270 uL of basophile medium from the aliquotes prepared in step 2 to each of the milk 2-5 epindorph tubes.
   b.  Transfer 30 uL from tube "milk 1" to "milk 2".  VORTEX. 
   c.  Take 30 uL from "milk 2" and add to "milk 3."  ETC.  VORTEX after each step.



CFSE labelling of 7day culture cells

  1. Suspend PBMCs at 10x106 cells/mL in PBS alone.
  2. Ensure that cells are uniformly suspended when CFSE is added.
  3. If CFSE negative control is needed, remove cells now.
  4. Make '2X' concentration (10 μM) in PBS
    • For example: add 5 mL PBS + 10 μL 5 mM CFSE
  5. Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
  6. Place in 37°C H2O bath x 10 min.
  7. Wash in 10 mL complete medium.
  8. Resuspend in medium at desired density


Proliferation Assay

Discussion

discuss this protocol

References

  1. Lyons AB and Parish CR. . pmid:8176234. PubMed HubMed [Lyons]
  2. Fuss IJ, Kanof ME, Smith PD, and Zola H. . pmid:19347849. PubMed HubMed [Fuss]
  3. Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn A, and Sampson HA. . pmid:19130927. PubMed HubMed [Shreffler]
All Medline abstracts: PubMed HubMed

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