User:Lara S. Ford/Notebook/Milk Study/2009/08/10

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Aug 7 2009 BM057-0 8-4-09 T Reg Studies (Day 3) Placed back in the incubator.
 * Divided cell culture wells
 * Mixed cells in each well by pipetting up and down several times.
 * Transferred 250 uL of cells in medium to 3 new wells labeled for each condition; this leaves 250 uL of cells in medium in the original wells.
 * Added 750 uL of AIM-V to each well to bring the total volume per well to 1 mL.

Aug 6 2009 (Clinic Day) Pulled charts to clarify questions/inconsistencies from eRAP data

BM057-0 8-4-09 48 Hour Culture (Day 2) BM058-0 8-3-09 T Reg Studies (Day 3) BM059-0 8-5-09 Basophil Activation (Day 1) BM060-0 8-5-09 Basophil Activation (Day 1)
 * Performed as per protocol by Alyssa Chase
 * Cell culture wells divided and fed by Alyssa Chase
 * Basophils acquired by flow cytometry from yesterday's experiments by Alyssa Chase

Aug 5 2009 BM058-0 8-3-09 48 Hour Culture (Day 2)


 * Cells from the 48 hour culture were harvested, processed as per the CD25 Selection and RNA Extraction protocol and stored in cluster tubes at -80.
 * One sample per stimulant condition:
 * Stimulant 1: caseins
 * Stimulant 2: medium alone
 * Stimulant 3: beads
 * Stimulant 4: egg white
 * Supernatants from the 48 hour culture were saved in cluster tubes and stored at -80.
 * One sample per stimulant condition:
 * Stimulant 1: caseins
 * Stimulant 2: medium alone
 * Stimulant 3: beads
 * Stimulant 4: egg white

BM059-0 8-5-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) BM060-0 8-5-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) (Specimens processed with Alyssa Chase) BM059-0: 2 green tops obtained containing approximately 9mL each for a total volume of 18mL of whole blood. BM060-0: 2 green tops obtained containing approximately 9mL each for a total volume of 18mL of whole blood.


 * Set aside 3 mL of whole blood for Basophil Activation assay.
 * Performed basophil activation as per protocol with the following deviation described by Alyssa for BM059-0: Tube A (RPMI + blood) initially contained staining buffer. To correct, I took all remaining blood and added RPMI to dilute 1:1. The total volume was about 1/2 of that called for in the protocol
 * Stored basophils in 5mL polypropylene tubes at 4C until the following day for flow.
 * Ficoll-separated remaining PBMCs:
 * BM059-0: 3.6 x 10^7 PBMCs (automated counter) --> 36 x 10^6 PBMCs.
 * BM060-0: 3.9 x 10^7 PBMCs (automated counter) --> 39 x 10^6 PBMCs.
 * Set aside 10 x 10^6 PBMCs for 48 hour culture.
 * Did not CFSE-label these cells.
 * Plated 2 x 10^6 CD25+ PBMCs in each of the following four conditions: AIM-V (medium alone, +CD3/CD28 beads, +caseins [alpha, beta, kappa], + egg white).
 * Set aside 10 x 10^6 PBMCs for 7 day culture.
 * CFSE-labeled these cells.
 * Resuspended cells in AIM-V at 4 x 10^6 cells/mL for the 7 day culture.
 * Plated 2 x 10^6 CD25+ PBMCs in each of the following four conditions: AIM-V + IL-2 (medium alone, +CD3/CD28 beads, +caseins [alpha, beta, kappa], + egg white).
 * Both cultures placed in incubator.
 * 48 hour culture will be removed on 8-7-09.
 * 7 day culture will be removed and harvested on 8-12-09.

Aug 4 2009 BM057-0 8-4-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) (Specimen processed with Alyssa Chase) BM057-0: 2 green tops obtained containing approximately 9mL each for a total volume of 18mL of whole blood.


 * Set aside 3 mL of whole blood for Basophil Activation assay.
 * Performed basophil activation as per protocol
 * Plated basophils as per protocol for same-day flow.
 * Ficoll-separated remaining PBMCs:
 * 3.6 x 10^7 PBMCs (automated counter) --> 36 x 10^6 PBMCs.
 * Set aside 10 x 10^6 PBMCs for 48 hour culture.
 * Did not CFSE-label these cells.
 * Plated 2 x 10^6 CD25+ PBMCs in each of the following four conditions: AIM-V (medium alone, +CD3/CD28 beads, +caseins [alpha, beta, kappa], + egg white).
 * Set aside 10 x 10^6 PBMCs for 7 day culture.
 * CFSE-labeled these cells.
 * Resuspended cells in AIM-V at 4 x 10^6 cells/mL for the 7 day culture.
 * Plated 2 x 10^6 CD25+ PBMCs in each of the following four conditions: AIM-V + IL-2 (medium alone, +CD3/CD28 beads, +caseins [alpha, beta, kappa], + egg white).
 * Both cultures placed in incubator.
 * 48 hour culture will be removed on 8-6-09.
 * 7 day culture will be removed and harvested on 8-11-09.
 * Acquired basophils by flow cytometry

BM058-0 8-3-09 Basophil Activation (Day 1)
 * Acquired basophils by flow cytometry from yesterday's experiment

Aug 3 2009 BM058-0 8-3-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) BM058-0: 2 green tops obtained containing approximately 9mL each for a total volume of 18mL of whole blood. (Specimen processed with Alyssa Chase)
 * Set aside 3 mL of whole blood for Basophil Activation assay.
 * Basophil activation performed as per protocol, with the following deviation described by Alyssa: Error during preparation of Ab cocktail: 110 uL of Ab alone were initially added to test tubes B-G (rather than Ab:Staining buffer). We then added enough staining buffer to these tubes to maintain the protocol's ratio of Ab:Staining buffer. The normal protocol was followed for tubes I-K.
 * Stored basophils in 5mL polypropylene tubes at 4C until the following day for flow.
 * Ficoll-separated remaining PBMCs:
 * 1.8 x 10^7 PBMCs (automated counter) --> 18 x 10^6 PBMCs.
 * Set aside 10 x 10^6 PBMCs for 48 hour culture.
 * Did not CFSE-label these cells.
 * Plated 2 x 10^6 CD25+ PBMCs in each of the following four conditions: AIM-V (medium alone, +CD3/CD28 beads, +caseins [alpha, beta, kappa], + egg white).
 * Set aside 10 x 10^6 PBMCs for 7 day culture.
 * CFSE-labeled these cells.
 * Resuspended cells in AIM-V at 4 x 10^6 cells/mL for the 7 day culture.
 * Plated 2 x 10^6 CD25+ PBMCs in each of the following four conditions: AIM-V + IL-2 (medium alone, +CD3/CD28 beads, +caseins [alpha, beta, kappa], + egg white).
 * Both cultures placed in incubator.
 * 48 hour culture will be removed on 8-5-09.
 * 7 day culture will be removed and harvested on 8-10-09.