Protocols/Template

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Overview

This protocol is designed for T-regulatory assay for Mouse allergen (Mus m1).

Materials

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.

• supply 1 (i.e. tubes of a certain size? spreaders?)
• reagent 1
• X μL reagent 2
  • component A (reagent 2 is made up of multiple components)
  • component B
• equipment 1
• equipment 2

==Procedure==

Isolation of Mononuclear Cells

• This is a sterile procedure and all steps should be performed in a hood.

1. Turn on the hood. Bring Ficoll and PBS to room temperature in the hood.
2. Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subjects information, i.e. ID#, date collected, date received.
3. If performing Basophil Activation Assay on this sample, set aside 3mL of whole blood before the next step.
4. Dilute the remaining blood at 1:1 with PBS in a 50mL conical tube.
5. 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.
6. Centrifuge @ 500g for 30minutes @ room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
7. 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 to bring the

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.
3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.