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(Isolation of Mononuclear Cells)
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==Overview==
==Overview==
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This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.
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Replace this sentence with a brief description of the protocol and its goal.
==Materials==
==Materials==
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List reagents, supplies and equipment necessary to perform the protocol here.  For those materials which have their own OWW pages, link to that page.  Alternatively, links to the suppliers' page on that material are also appropriate.
 
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*supply 1 (i.e. tubes of a certain size? spreaders?)
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For a 50 μL PCR reaction:
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*reagent 1
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*X &mu;L reagent 2
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**component A (reagent 2 is made up of multiple components)
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**component B
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*equipment 1
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*equipment 2
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=='''Procedure'''==
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* 35 μL H<sub>2</sub>O
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* 5 μL 10X PCR buffer
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* 5 μL 2mM dNTPs (each)
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* 1.5 μL 50mM MgCl<sub>2</sub>
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* 1 μL 50μM sense primer
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* 1 μL 50μM antisense primer
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* 1 μL 5nM DNA template
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* 0.5 μL TAQ DNA polyermerase
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* This is a sterile procedure and all steps should be performed in a hood.
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==Procedure==
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# Turn on the hood.  Bring Ficoll and PBS to room temperature in the hood.
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# In a PCR tube, mix the components on ice in the order they are listed above.
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# Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subjects information, i.e. ID#, date collected, date received.
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# Perform thermocycling program
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# If performing '''Basophil Activation Assay''' on this sample, set aside 3mL of whole blood before the next step.
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## 95 °C 5 min
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# Dilute the remaining blood at 1:1 with PBS in a 50mL conical tube.
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## 95 °C 30 s
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# Place 15mL of Ficoll in a 50mL conical tube.  Overlay with up to 30mL of diluted blood, gently add the blood on the ficoll solution to avoid of blood with ficoll solution.
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## T<sub>H</sub> 30 s
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# Centrifuge @ 500g for 30minutes @ room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
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## 72 °C 1 min for each 1 kb PCR product
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# Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50mL conical tube (avoid aspirating the ficoll).  Add PBS into the 50mL tube to bring the sample to a minimum of 2X the initial volume.  Invert up and down gently to mix.
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## Repeat steps 2-4 a total of 12-36 times (24 is standard).
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# Centrifuge @ 500g for 20minutes @ room temperature (maximum acceleration and deceleration).
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## 72 °C 5 min
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# Aspirate and discard the supernatant.  Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding 1mL of PBS.  Set aside a 10μl aliquot of cells for counting as follows:  Add 90μl of PBS into the 10μl of cells.
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## 12 °C hold
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# Add PBS to the cells in the 50mL tube for a total volume of 20mL, and centrifuge @ 300g for 15 minutes @ room temperature(maximum acceleration and deceleration).
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# In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.  Combine the 100μL aliquot of cells in PBS set aside above with 100μL of 0.2% Trypan solution (if using the automated counter) or 0.4% Trypan solution (if manual counting).  Mix well with pipette.
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# Carefully, introduce 10μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter.  Count the number of cells under a microscope or place 20μL of the stained cells onto a disposable slide and count using the automated counter.
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# After centrifugation is completed, aspirate and discard the supernatant.  Resuspend the cell pellet by tapping the tube until no clumps are visible.  Suspend PBMCs with PBS @ 10 million cells/mL in a 15mL conical tube.
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=='''PBMC Antigen Stimulation Assay'''==
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==*This is a sterile procedure and all steps should be performed in a hood.==
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# Remove stimulants from the freezer and thaw.
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# Label 24 well plate with specimen ID and date (this is for the 7 days cell culture).  Label each well with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).
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## Musm1 (allergen):  @ 200μg/mL purified Musm1 protein in Aim-V.
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## AIM-V + IL-2 (negative control): AIM-V + IL-2 medium alone.
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## Beads (positive control): 1μg/mL anti-Cd3/Cd28 beads.
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## Tetanus:  200μg/mL tetanus in AIM-V.
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# Add an equal volume (1:1 dilution) of freshly prepared 10μM CFSE (in PBS) to the tube of cells.  To make 1.5mL of PBS + CFSE (2x solutin): add 3μL of stock CFSE (5mM) into 1.5mL of PBS.
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# Incubate in 37deg;C water bath for 10 minutes.
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# After incubation, wash the CFSE stained cells in 10mL of AIM-V @ 300g for 10 minutes.  Aspirate supernatant after centrifugation.
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# Resuspend CFSE stained cells in AIM-V medium @ 4 million cells/mL.  For plating, each well should contain 2 milliion cells/mL.
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*  Begin the stimulation process by preparing AIM-V medium + a 2X solution of IL-2 by adding 2μL of IL-2 per mL of medium in a 15mL conical tube.  For 5 stimulant conditions, you will need atleast 2.5mL of AIM-V + IL-2.  Vortex gently.
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==Notes==
==Notes==
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==References==
==References==
'''Relevant papers and books'''
'''Relevant papers and books'''
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<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
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<!-- If this protocol has papers or books associated with it, list those references here.-->
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<!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently.
<biblio>
<biblio>
#Goldbeter-PNAS-1981 pmid=6947258
#Goldbeter-PNAS-1981 pmid=6947258
#Jacob-JMB-1961 pmid=13718526
#Jacob-JMB-1961 pmid=13718526
#Ptashne-Genetic-Switch isbn=0879697164
#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>
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</biblio>-->
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<!-- Try the [[Template:FormatRef|FormatRef template]]-->
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#{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258
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#{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526
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#{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164
==Contact==
==Contact==

Current revision

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Contents

Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

For a 50 μL PCR reaction:

  • 35 μL H2O
  • 5 μL 10X PCR buffer
  • 5 μL 2mM dNTPs (each)
  • 1.5 μL 50mM MgCl2
  • 1 μL 50μM sense primer
  • 1 μL 50μM antisense primer
  • 1 μL 5nM DNA template
  • 0.5 μL TAQ DNA polyermerase

Procedure

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform thermocycling program
    1. 95 °C 5 min
    2. 95 °C 30 s
    3. TH 30 s
    4. 72 °C 1 min for each 1 kb PCR product
    5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
    6. 72 °C 5 min
    7. 12 °C hold

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
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References

Relevant papers and books

  1. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
  2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
  3. Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164

Contact

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