User:Lara S. Ford/Notebook/Milk Study/Entry Base: Difference between revisions

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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Baked Milk 2</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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<h2> Aug 7 2009 </h2>
<h4> BM057-0 8-4-09 T Reg Studies (Day 3) </h4>
* 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.
Placed back in the incubator.
<h2> Aug 6 2009 (Clinic Day) </h2>
Pulled charts to clarify questions/inconsistencies from eRAP data
<h4> BM057-0 8-4-09 48 Hour Culture (Day 2) </h4>
* Performed as per protocol by Alyssa Chase
<h4> BM058-0 8-3-09 T Reg Studies (Day 3) </h4>
* Cell culture wells divided and fed by Alyssa Chase
<h4> BM059-0 8-5-09 Basophil Activation (Day 1) </h4>
<h4> BM060-0 8-5-09 Basophil Activation (Day 1) </h4>
* Basophils acquired by flow cytometry from yesterday's experiments by Alyssa Chase
<br>
<h2> Aug 5 2009 </h2>
<h4> BM058-0 8-3-09 48 Hour Culture (Day 2) </h4>
* 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
<br>
* 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
<br>
<h4> BM059-0 8-5-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) </h4>
<h4> BM060-0 8-5-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) </h4>
<p>(Specimens processed with Alyssa Chase)</p>
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.
<br>
* 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.
<br>
* 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).
<br>
* 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).
<br>
* 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.
<br>
<h2> Aug 4 2009 </h2>
<h4> BM057-0 8-4-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) </h4>
<p>(Specimen processed with Alyssa Chase)</p>
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.
<br>
* Ficoll-separated remaining PBMCs:
** 3.6 x 10^7 PBMCs (automated counter) --> 36 x 10^6 PBMCs.
<br>
* 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).
<br>
* 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).
<br>
* 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.
<br>
* Acquired basophils by flow cytometry
<br>
<h4> BM058-0 8-3-09 Basophil Activation (Day 1) </h4>
* Acquired basophils by flow cytometry from yesterday's experiment
<br>
<h2> Aug 3 2009 </h2>
<h2> Aug 3 2009 </h2>
<h4> BM058-0 8-3-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) </h4>
<h4> BM058-0 8-3-09 Basophil Activation / 48h Hour Culture / T Reg Studies (Day 0) </h4>
BM058-0: 2 green tops obtained containing approximately 9mL each for a total volume of 18mL of whole blood.  
BM058-0: 2 green tops obtained containing approximately 9mL each for a total volume of 18mL of whole blood.  
 
<p>(Specimen processed with Alyssa Chase)</p>
* Set aside 3 mL of whole blood for Basophil Activation assay.  
* Set aside 3 mL of whole blood for Basophil Activation assay.  
** Basophil activation performed by Alyssa Chase as per protocol, with the following deviation: 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.
** 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.  
** Stored basophils in 5mL polypropylene tubes at 4C until the following day for flow.  
<br>
<br>

Revision as of 10:06, 10 August 2009

Baked Milk 2 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page

Aug 7 2009

BM057-0 8-4-09 T Reg Studies (Day 3)

  • 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.

Placed back in the incubator.


Aug 6 2009 (Clinic Day)

Pulled charts to clarify questions/inconsistencies from eRAP data


BM057-0 8-4-09 48 Hour Culture (Day 2)

  • Performed as per protocol by Alyssa Chase

BM058-0 8-3-09 T Reg Studies (Day 3)

  • Cell culture wells divided and fed by Alyssa Chase

BM059-0 8-5-09 Basophil Activation (Day 1)

BM060-0 8-5-09 Basophil Activation (Day 1)

  • 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.
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