Shreffler:Milk Program

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(Procedure)
(Procedure)
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#Incubate tubes for 30 minutes at 37°C (in incubator).   
#Incubate tubes for 30 minutes at 37°C (in incubator).   
#Prepare the cocktail. Label a 5 mL polypropylene tube "staining cocktail"  Add 700 uL of staining buffer.
#Prepare the cocktail. Label a 5 mL polypropylene tube "staining cocktail"  Add 700 uL of staining buffer.
-
a. 35 uL of HLA-DR-PE-Cy7 mAb
+
    a. 35 uL of HLA-DR-PE-Cy7 mAb
-
b. 70 uL of CD63- FITC
+
    b. 70 uL of CD63- FITC
-
c. 70 uL of CD203c-PE
+
    c. 70 uL of CD203c-PE
-
d. 70 uL of CD123 PE-cy5
+
    d. 70 uL of CD123 PE-cy5
-
e. 70 uL of CD41a
+
    e. 70 uL of CD41a
-
f.  70 uL of CD3
+
    f.  70 uL of CD3
-
g. 70 uL of CD14
+
    g. 70 uL of CD14
-
h. 70 uL of CD19-APC
+
    h. 70 uL of CD19-APC
#
#
#
#

Revision as of 17:46, 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
  2. Bring 15/10mL of Ficoll(sterile) to room temperature and put 5mL RPMI in water bath.
  3. Collect pre-made stimulants from the freezer (Rm 40 in clear boxes with rubber bands). LABEL each one
  4. 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.
  5. Label the 5 remaining tubes "Basophil medium." Add 30 uL of the cocktail into these tubes.
  6. Add 270 uL of the warmed RPMI to the five basophil tubes(made above), fMLP, milk extract, and anti-IgE.
  7. Add 135 uL of RPMI to PMA and CaI and then combine them.
  8. Prepare a 10-fold dilution of milk stimulants:
   a.  Transfer 270 uL of basophil 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.
  1. Label 5 mL polypropylene tubes A-K. Remember to include subject number on tube A as BM055-06.
             list of stimulants
  1. Transfer 250 uL of warm RPMI to tubes A & B.
  2. Transfer 250 uL of each stimulant to the appropriate polypropolene tube (C-K) and keep them in incubator until ready to start.
  1. Get the blood from the clinic, open the two green top tubes under the hood.
  2. (keep sterile) Set aside 3mL of blood into pre-labeled tube for basophil assay.
  3. Transfer remaining blood in pre-labeled 50mL tube and dilute 1:1 with PBS
  4. Overlay with 30 mL of diluted blood on the 15mL ficoll.
  5. Centrifuge at 500 g for 30 minutes at 23°C with acceleration slow and brake off.

now back to basophil protocol

  1. Take out the labelled tubes from incubator
  2. Transfer 250 uL of participant blood to each tube A-K.
  3. Incubate tubes for 30 minutes at 37°C (in incubator).
  4. Prepare the cocktail. Label a 5 mL polypropylene tube "staining cocktail" Add 700 uL of staining buffer.
   a. 35 uL of HLA-DR-PE-Cy7 mAb
   b. 70 uL of CD63- FITC
   c. 70 uL of CD203c-PE
   d. 70 uL of CD123 PE-cy5
   e. 70 uL of CD41a
   f.  70 uL of CD3
   g. 70 uL of CD14
   h. 70 uL of CD19-APC


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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