Milk allergy is the most common cause of food allergy in infants and young children, affecting 2.5% of infants in industrialized countries. Previous studies suggest that the majority will tolerate heat-denatured products without harmfμL effects. Our preliminary findings that ingestion of extensively baked-milk products may enhance tolerance induction. This study is designed to determine whether frequent dose escalation of milk protein in allergic patients woμLd increase the tolerance of their immune system.
Through the baked milk 2 study, we plan to study the immunologic mechanism associated with oral tolerance induction. We propose to investigate the effect of introducing baked-milk products by comparing the rate of tolerating less heated milk in children subjected to more frequent dose-escalation (every 6 months) and in children subjected to less frequent maintenance increases (every 12 months).There will be a comparison of the tolerance of an individual patient longitudinally over a period of time. Also, the changes in T regμLatory cells woμLd be evaluated in five groups, who are given different doses of baked milk proteins.
Visit 1 Procedures
Isolation of PBMC's, Basophil Activation Assay, CFSE Labeling, T RegμLatory Cell Assay, & Antigen Activation Assay
Isolation of PBMC's (General protocol described ____)
- 18 mL sample of whole heparinized blood/patient
- Ficoll Paque Plus [endotoxin tested], room temperature
- Sterile phosphate buffered saline (PBS), room temperature
- 0.2% and 0.4% Trypan Solution
- Sterile conical tubes (15 mL, 50 mL)
- Sterile, graduated transfer pipettes
- Hemocytometer or disposable slides for the automated counter
Basophil Activation Assay (General protocol described: ___)
- 3 mL sample of whole herparinized blood/patient
- RPMI mediμM (store at 4°C in dark)
- 1x FACS Lysing Solution (made from 10X stock w/dH2O, store at 4°C, expires in 1 week)
- Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)
- Monocolonal Antibody cocktail (CD63-FITC, CD123 PE-Cy5, HLA-DR-PE Cy7, CD41a-APC, CD3-APC, CD14, CD19-APC) (store at 4°C in the dark).
- Polypropylene tubes
- Pre-made 30 μL aliquots (stored at -30°C):
a. Basophil mediμM w/ 20 μM of fMLP b. Basophil mediμM w/ 200 μg/mL milk protein c. Basophil mediμM w/ 20 μg/mL anti-IgE d. Basophil mediμM w/ 20X PMA (5 μg/mL ) e. Basophil mediμM w/ 20X CalciμM Ionophore (20 μg/mL)
- Sample: 10x10^6 cells
- Phosphate buffered saline (PBS)
- CFSE (5 μM aliquots)
- AIM-V Media (add 500 μL amphotericin (fungizone), 5 mL PS (PCN-sterptomycin), 5 mL glutamine)
PBMC Antigen StimμLation Assays
- Samples: 10 x 10^6 ce;;s fpr 48 hours cμLture for measurement of Th1/Th2 gene transcription in allergen-activated cells by RT-PCR AND 10 x 10^6 cells for 7 day cμLture (for measurement of frequency of PBMC-derived milk-specific CD4+ CD25+ FoxP3+ T cells).
- AIM-V Media [add 500μL amphotericin (fungizone), 5 mL PS (PCN-streptomycin), 5mL gluatmine]
- IL-2 (approximately 20 units/μL; 1 μg = 2.4 x 10^3 units) [To make a 10μg vial of IL-2, reconstitute in 1 mL sterile PBS + 1% hμMan AB serμM to get a final concentration of 10μg/mL. Place in aliquots of 50 μL and freeze at -80°C.
- Obtain CD3, CD28 expander beads (can be foundi n the refrigerator in the white "milk project" box in Rm 46.)
- Caseins (alpha, beta, and kappa) (20 mg/mL).
- Egg white (stock 2 mg/mL)
- 24 well tissue cμLture plates
- Sterile 5 mL polyproylene round bottom tubes
Basophil Assay and Isolation of Mononuclear Cells (done simμLtaneously)
- 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.
- Collect pre-made stimμLants 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 μL RPMI + 3.6 μL IL-3. MIX WELL.
- Label the 5 remaining tubes "Basophil mediμM." Add 30 μL of the cocktail into these tubes.
- Add 270 μL of the warmed RPMI to the five basophil tubes(made above), fMLP, milk extract, and anti-IgE.
- Add 135 μL of RPMI to PMA and CaI and then combine them.
- Prepare a 10-fold dilution of milk stimμLants:
- Transfer 270 μL of basophil mediμM from the aliquots prepared in step 2 to each of the milk 2-5 epindorph tubes.
- Transfer 30 μL from tube "milk 1" to "milk 2". VORTEX.
- Take 30 μL from "milk 2" and add to "milk 3" etc. Vortex after each step.
9. Label 5 mL polypropylene tubes A-K. Remember to include subject nμMber on tube A. list of stimμLants
10. Transfer 250 μL of warm RPMI to tubes A & B.
11. Transfer 250 μL of each stimμLant to the appropriate polypropolene tube (C-K) and keep them in incubator until ready to start.
12. Get the blood from the clinic, open the two green top tubes under the hood.
13. (keep sterile) Set aside 3mL of blood into pre-labeled tube for basophil assay.
14. Transfer remaining blood in pre-labeled 50mL tube and dilute 1:1 with PBS
15. Overlay with 30 mL of diluted blood on the 15mL ficoll.
16. Centrifuge at 500 g for 30 minutes at 23°C with acceleration slow and brake off. now back to basophil protocol
17. Take out the labeled tubes from incubator
18. Transfer 250 μL of participant blood to each tube A-K.
19. Incubate tubes for 30 minutes at 37°C (in incubator).
20. Prepare the cocktail. Label a 5 mL polypropylene tube "staining cocktail" Add 700 μL of staining buffer.
a. 35 μL of HLA-DR-PE-Cy7 mAb b. 70 μL of CD63- FITC c. 70 μL of CD203c-PE d. 70 μL of CD123 PE-cy5 e. 70 μL of CD41a f. 70 μL of CD3 g. 70 μL of CD14 h. 70 μL of CD19-APC
21. Remove tubes from incubator and add 50 μL cold PBS w/20 mM EDTA to each tube to stop degranμLation.
22. Stain cells by adding 110 μL of the prepared Ab Cocktail to tubes B-K. Do NOT add to Tube A.
23. Put it in the refrigerator(4°C) for 30 minutes.
- Return to isolation of PBMC's. Remember to keep conditions STERILE!
24. Remove ficoll tubes from centrifuge. Using a sterile transfer pipette , collect PBMCs (cloudy gray layer) into a new 50 mL conical tube.
25. Under the hood, add sterile PBS to double the volμMe and invert the tube to mix. Centrifuge at 500 g for 20 minutes at room temperature (max acceleration and deceleration).
26. Aspirate and discard the supernatant. Resuspend the pellet by tapping the tube until no clμMps are visible, then adding 1 mL of PBS.
27. Set aside a 10 μL aliquot of cells for counting: 10 μL of cells into 90 μL of PBS in a STERILE eppendorf tube.
28. Add PBS to cells to make a total volμMe of 20 mL.
29. Centrifuge at 300 g for 15 minutes at room temperature (maximμM acceleration and deceleration).
30. While the cells are in the centrifuge, you must determine the volμMe to use for resuspending the PBMCs after this wash. This in turn requires knowledge of the total nμMber of cells in the sample:
a. Combine the 100 μL aliquot of cells in PBS set aside in Step 28 with 100 μL of 0.2% Trypan solution (if using the automated counter) or 0.4% Trypan soplution (if manual counting).
b. Mix well with a pipette.
c. Place 20 μL of the stained cells onto a disposable slide.
e. To take the cell count, in the computer program, write dilution as a whole nμMber (20x). [1st diluted 10 μL of cells in 100 μL total volμMe PBS + Cells, then added an additional 100 μL of Trypan solution
= 10μL/200μL = 1/20 = 20 fold dilution).
f. Choose cell type from drop down menu ("HμMan")
g. Pick "display"
h. Focus image so the cells appear yellowish
i. Hit "Count."
31. After centriguation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clμMps are visible. Suspend PBMCs at 10 x 10^6 cells/mL in PBS. To calcμLate this: Take nμMber counted in step 31i divided by 10 x 10^6 cells/mL. This gives you the the total volμMe of PBS you must add to the cells.
32. Divide Cells as follows: a. 10 x 10^6 cells for measurement of Th1/Th2 gene transcription in allergen-activated cells by RT-PCR (after 48 hr cμLture). b. 10 x 10^6 cells for measurement of frequency of PBMC-derived, milk-specific CD4+, CD25+, FoxP3+ T cells (after 7 day cμLture). c. If indicated, remaining cells will undergo CD25 Depletion Experiments.
- Note, if you collect less than 2 mL of PBMC's in step 32, just divide the volμMe equally to the 48 hour and 7 day test tubes in step 33.
33. In a 15 mL conical tube, make a PBS: CFSE solution: a. 1 mL PBS b. 2 μL of 5 μM CFSE
34. Add the PBS: CFSE solution just prepared to the 7 DAY CμLTURE PBMC's.
35. Place in a 37°C water bath for 10 minutes.
PBMC Antigen StimμLation Assay
36. STERILE CONDITIONS. Label two 24 well plates/their lids with specimen ID and date. Label each well with the appropriate condition, ordered by priority (for cases where there are insufficient cells to test all stimμLants). a. Allergens- Caseins (C+): 50 μg/mL purified casein proteins (alpha, beta, kappa) in AIM-V mediμM b. Negative Control- AIM-V mediμM (A+): MediμM alone c. Positive Control- Beads (B+): 1 μg/mL anti-CD3, anti- CD28 beads in AIM-V d. Egg white (E+): 20 μg/mL egg white in AIM-V
37. Label one plate for 48 hour cμLture and a second plate for a 7 day cμLture.
38. Add 10 mL of AIM-V to both 7 and 48 hour cμLture tubes and spin at 300 g for 10 minutes at room temperature.
39. STERILE CONDITIONS: Aspirate the cells.
40. Under the hood, resuspend cells in 2.5 mL of AIM-V mediμM to obtain a concentration of 4 x 10^6 cells/mL (For plating, each well shoμLd contain at 2-2.5 x 10^6 cells and a total volμMe of 1 mL).
41. Prepare solutions for each stimμLant condition in sterile, 5 mL polypropyene tubes. Label each tube with a notation for 48 hour cμLture and A+, B+, C+, E+. Also, prepare tubes for the 7 day cμLture samples and also label A+, B+....
42. 48 Hours Antigen StimμLation Preparation a. Place 500 μL of AIM-V in each of the test tubes. b. Add 500 μL of the 48 hr-labeled cells in AIM-V to each tube and mix by pipetting up and down. c. Then add to the respective tubes:
A+: MediμM Alone. Do not add any other substances.
B+: Add 5 μL of CD3, CD28, being sure vortex first to resuspend the beads in their container.
C+: Add 2.5 μL of EACH casein (alpha, beta, kappa).
- Be sure to mix by pippetting up and down.**
43. 7 Day Antigen StimμLation Preparation: Unlike 48 hour cμLture, this contains CFSE labeled cells and IL-2.
a. Create a cocktail. Place 2 mL of AIM-V + 4 μL of IL-2 in an appropriately labeled test tube. Vortex gently. b. Add 500 μL of the cocktail to the labeled A+, B+, etc. test tubes. c. To each tube, add 500 μL of the 7 day cells set aside in step 33. Mix. d. Then add to the respective tubes:
A+: AIM-V mediμM + IL-2 alone. Do not add any other substances.
B+: Add 5 μL of CD3, CD28 expander beads. Be sure to vortex the beads in their container. Then suspend the beads in the AIM-V/IL-2 solution by pipetting up and down.
C+: Add 2.5 μL of each casein (alpha, beta, kappa). Pipette up and down.
E+: Add 10 μL of egg white. Pipette up and down.
44. Plate the stimμLants for both the 48 hr and 7 day samples into the wells labeled in step 37. Then place the tissue cμLture plate in the incubator.
Procedure for Splitting Cells with 7 Day Incubation Period
If you receive a sample on Monday or Tuesday, splitting shoμLd be completed on Friday. For Wednesday samples, split on Monday.
1. Obtain the appropriate plate. Set pipette to 250 μL. Mix by pipeting up and down in each well.
2. Take 250 μL of cμLture from the well labeled A+ and add it to the three wells vertical to it. Repeat with conditions B+ through E+.
3. Add 750 μL of AIM-V at ROOM TEMPERATURE to each well.
48 Hours Protocol
1. Obtain the plate labeled "48 hours" that was placed in the incubator during the Day 0 procedure. Aliquot 30 mL of running buffer to be used throughout the experiment.
2. Label four 5 mL polystyrene tubes (A+, B+, etc.). Remember to have the ID of the patient on the first of the tubes. Collect the specimen from the incubator dated two days before the date the 48 hr procedure is performed.
3. Label 8 CLUSTER TUBES as follows:
a. Specimen ID
b. Date of original cμLture
c. 4 tubes-- Supernatants, 48 hr. AND 4 tubes-- Cx Cells, 48 hr.
d. A+, B+, C+, E+
4. Set pipette to 1000 µL. Resuspend cells by pipetting up and down (getting the "four corners" of the well), and then placing the fluid in their respective tubes. Be sure to transfer the total volume/well.
5. Centrifuge tubes at 300 g for 5 minutes at room temperature.
6. Transfer 800 μL of supernatant from the culture tube into each corresponding cluster tube. Be careful not to disturb the cell pellets.
7. Cap the cluster tubes and store in the -80 C freezer. Keys for the freezer are on a blue chain by the lab bench. Our box is in the top fridge, bottom right, and it is labeled "milk project."
8. Shake tube gently to dissolve pellet.
9. Add 1 mL of running buffer (a type of Cell Separation Buffer) to the dissolved pellet tubes.
10. Centrifuge at 300 x g for 5 minutes. Aspirate.
11. Resuspend cells in 80 μL of running buffer.
12. Obtain anti-CD25 Microbeads from the small yellow and blue box from the fridge in Rm 46.
13. IN THE HOOD add 10 μL of anti-CD25 Microbeads to each sample.
14. Mix well and incubate for 15 minutes in the refrigerator.
15. Cool the Centrifuge to 4°C (but do not spin anything).
16. Wash cells by adding 1.5 mL of running buffer and centrifuge at 300 g for 10 minutes at 4˚C.
17. Aspirate supernatant completely.
18. Resuspend cells in 500 μL of cell separation buffer. (PAY ATTENTION TO UNITS!!)
19. Obtain the green and black magnet (OctoMACS magnet) from the large cabinet under the bench.
20. Obtain Macs separation colμMns (Also called MS colμMns) from the supply station in the back of Rm 46. Be sure to keep colμMns on paper towels, not on the bench.
21. Place one MS colμMn for each of the give cell cμLtures onto the OctoMACS magnet.
22. Underneath the columns, place 5mL polypropylene tubes.
23. Flush each column with 500 μL of running buffer.
24. Apply cell suspensions to the corresponding columns.
25. Add 1 mL of running buffer to the original tubes to wash.
26. Once the column reservoir is empty, remove 500 μL of buffer from the original tubs and apply to the colμMns 2 times. (500 μL each time)
27. Remove columns from the magnet and place tip into an appropriately labeled eppendorf tube.
28. Pipette 1 mL of buffer into the column.
29. IMMEDIATELY flush out the fraction with magnetically-labeled CD25+cells by firmly applying the plunger supplied with the colμMn. To do this, hold the eppendorf tube and bottom of colμMn in left hand. Make sure injector is far from the bottom of the tube to avoid flood. Then tighten on the plunger. When you push, make sure to give the fluid room to flow.
30. Using a MICROCENTRIFUGE collect the pellets. Turn the eppendorf tubes so that the opening is facing the middle of the centrifuge. Be sure to balance.
31. Microcentrifuge at 400 g for 5 minutes.
32. BE CAREFUL!! Using suction and a 20 μL pipette tip, aspirate most of the supernatant. Since the supernatant has collected on the back of the tube, make sure you position the pipette tip so that it sucks from the front of the tube.
33. Resuspend cells in 100 μL of RLT buffer containing B-mercaptoethanol (in large cabinet under the bench). Resuspend the cells by pipetting up and down 10x and rinsing the walls carefμLly.
34. Vortex each eppendorf tube twice for 15 s each time to collect all the liquid at the bottom of the tube.
35. Transfer to appropriately labeled cluster tubes and freeze at -80 C.
== 7 Day Protocol ==
1. Cool centrifuge at 4°C. Portion 30 mL of staining buffer in 50 mL conical tube.
2. Label 4 cluster tubes for 7 day cells and 7 day supernatants as following:
a. Specimen ID b. Date c. Supernatants- 48 hr or 7 day d. A+, B+, etc.
3. Collect the appropriate 24 well plate from the incubator. Label 4 polystyrene staining tubes corresponding to each well (A+, B+, etc).
4. Harvest cells from cμLture wells using a 1, 000 μL pipette by pipetting up and down to resuspend cells in the well and rinsing each well with 200 μL of staining buffer.
5. Centrifuge tubes at 300 x g for 5 minutes at 4˙C.
6. For each stimulant, using a 1000 μL pipette tip, transfer 800 μL of supernatant from the culture tube into each corresponding cluster tube. Be careful not to disturb their pellets. If you do, be sure to spin again.
7. Cap the cluster tubes and store in the -80°C freezer by the freight elevators.
8. Add 1 mL of staining buffer to each tube and vortex.
9. Wash cells at 300 g for 5 minutes at 4°C. Decant tubes.
10. Add 1 mL of staining buffer to each tube and vortex.
11. Wash cells at 300 g for 10 minutes at 4°C. Decant tubes.
12. Prepare cocktail preparation (enough for 4 tubes), obtaining the markers from the white box labeled "milk project" in the refrigerator in Rm. 46 near centrifuge. Be sure to store in the refrigerator (light sensitive) until ready for use.
a. 50 μL of CD25-PCy5 b. 25 μL CD4-PC7 c. 25 μL of CD3-APC7 d. 10 μL of Violet live/dead (2 μL), which can be collected from from the refrigerator in the back of Rm 46 near the lunch room. If you need to open a new box, be sure to dilute with 100 μL of DMSO and date and initial the tube. e. 5 μL of CD127-PE f. Add 135 µL of staining buffer to reach a total volμMe of 250 μL.
13. Add 50 μL of cocktail to each tube.
14. Place in the fridge for 20-30 minutes (This could be a good time to split cells).
15. Add 3 mL of staining buffer to each tube and vortex.
16. Wash at 300 g for 10 minutes at 4°C. Decant tubes.
17. Resuspend cells in 500 μL 1x FACS lysing solution and allow to stand for 15 minutes at room temperature in the dark.
18. IN THE HOOD, prepare a solution of 20% DMSO in staining buffer. Add 2 mL of staining buffer and 500 μL of 20% STERILE DMSO. Add 500 µL of 20% DMSO to each tube. Mix gently but thoroughly.
19. Transfer the samples to appropriately-labeled cluster tubes and store samples at -80°C.
(Done later to frozen 7day cells)
1. Thaw frozen samples.
2. Remove samples from cluster tubes and polystyrene
Vist 2 Protocols
Tolerance to Denatured Milk – CD25+ Depletion Manual of Procedures August 28, 2009
CD25 DEPLETION for first followup visit (at 6 or 12 months)
- 27 x 10^6 PBMC's
- 5μM CFSE - PBS - AIM-V - Robosep Buffer - EasySep Kit - EasySep Magnet
Purpose: Comparative 7 Day incubation of control PBMCs and CD25 (i.e. T-regulatory) depleted PBMCs.
- After counting ("Isolation of Mononuclear Cells" protocol), resuspend ALL PBMCs in PBS at 10 x 10^6 cells/mL.
- CFSE staining
- In a separate 15 mL conical tube, add the equivalent amount of PBS as used to resuspend the cells in step 1.
- Add 5 μM CFSE at a ratio of 2 μL of 5 μM CFSE/ 1 mL of PBS.
- Add this freshly prepared PBS:CFSE solution to the PBMCs.
- Place in a 37°C water bath for 10 minutes.
- Add 15 mL of AIM-V, spin at 300 g for 10 minutes at room temperature. Aspirate supernatant.
- CD25 Isolation
- Resuspend the pellet in Robosep Buffer to obtain a concentration of 50 million cells/mL. (i.e., for 30 million cells, add 600 μL).
- Add 1 million cells (i.e. 20 μL of above solution) to 1mL of PBS in a polystyrene tube labeled "CD25 pre." Set aside (for flow cytometry in 24h).
- Add 10 million cells (i.e. 200 μL) to 10mL AIM-V in a 15mL conical tube labeled "CD25 control." Set aside (for later plating - see step 34).
- Transfer remaining PBMCs into a 5 mL polysterene tube.
- Add EasySep positive selection cocktail at 25 μL/mL of cells.
- Mix well and incubate for 15 minutes.
- Mix EasySep Magnetic Nanoparticles to ensure that they are in uniform suspension by pipetting up and down vigorously, more than 5 times. DO NOT VORTEX.
- Add Nanoparticles at 50 μL/mL of cells.
- Mix well and incubate for 10 minutes.
- Use Robosep to bring total volume to 2.5 mL (half the height of 5mL tube). Mix cells in the tube by gently pipetting up and down 2-3 times. Place the tube without the cap into the magnet and set aside for 5 minutes.
- Pick up the magnet, and in one continuous motion invert the magnet and tube and pour the supernatant fraction into a 5mL polystyrene tube labeled "CD25-". Leave the magnet inverted for 2-3 seconds, then return to the upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube. Discard the tube in the magnet, which contains cells that have been positively selected for CD25.
- Centrifuge the supernatant at 300 x g for 10 minutes. Resuspend cells in 1 mL AIM-V.
- Add 10 μL of cells into a small eppendorf tube containing 25μL of PBS and 15 μL of 0.2% Trypan (to create a 1:5 dilution). Count using automated cell counter.
- Transfer contents of tube to a 15 mL conical tube labeled "CD25-". Add 10 mL AIM-V.
- Centrifuge the tubes labeled "CD25-" and "CD25 control" (set aside in step 9) at 300 x g for 10 minutes. Aspirate.
- Resuspend the 1 million cells in "CD25 Control" in 2.5 mL AIM-V to bring to a total concentration of 4x10^6 cells/mL.
- Count the "CD25-" cells: Resuspend in 1 mL AIM-V. Add 10 μL of cells into a small eppendorf tube containing 25μL of PBS and 15 μL of 0.2% Trypan (to create a 1:5 dilution). Count using automated cell counter. Resuspend the "CD25-" cells in AIM-V at a concentration of 4x10^6/mL.
- Set aside 1 million cells in 1mL of PBS in a polystyrene tube labeled "CD25- Post."
- Plate CD25 control and CD25- cells using the "PBMC Antigen Stimulation Assay" protocol for 7 day cells (i.e. CFSE labeled cells in AIM-V mediμM containing IL-2). If we counted less than 9x10^6 cells in step 32, we may need to eliminate certain conditions for the CD25- group. If this occurs, add stimilants according to priority: Caseins, AIM-V, Beads, Egg White.
- Place the cells in the incubator.
- Incubate cells at 37°C for 7 days, being sure to split when appropriate. Follow the standard "7 DAY PROTOCOL" at the end of the incubation period for both plates.
- Centrifuge the "CD25+ pre" and "CD25-post" tubes set aside in steps 8 and 33 at 300 x g for 5 minutes and resuspend in 1 mL of 1xFACS lysing buffer. Store at 4°C for 24 hours and then acquire using flow cytometry. This step is to check effectiveness of CD25 depletion.
- Lyons AB and Parish CR. . pmid:8176234.
- Fuss IJ, Kanof ME, Smith PD, and Zola H. . pmid:19347849.
- Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn A, and Sampson HA. . pmid:19130927.
- Sicherer SH and Sampson HA. . pmid:16455349.
- Sainte-Laudy J, Boumediene A, Touraine F, Orsel I, Brianchon C, Bonnaud F, and Cogné M. . pmid:17659434.
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare