Shreffler:Milk Program

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. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7. DOI:10.1016/0022-1759(94)90236-4 |
2. Fuss IJ, Kanof ME, Smith PD, and Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol. 2009 Apr;Chapter 7:7.1.1-7.1.8. DOI:10.1002/0471142735.im0701s85 |
3. Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn A, and Sampson HA. Association of allergen-specific regulatory T cells with the onset of clinical tolerance to milk protein. J Allergy Clin Immunol. 2009 Jan;123(1):43-52.e7. DOI:10.1016/j.jaci.2008.09.051 |

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare