Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Podsumowanie

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically ≥ 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Streszczenie

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are ≥ 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Protokół

Please read and follow the protocol included in the Miltenyi kit or online at http://www.miltenyibiotec.com/.

Dyskusje

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are ≥ 96% pure CD4+/CD3...

Materiały

Material Name	Type	Company	Catalogue Number	Comment
Name	Company	Catalog Number	Comments
MACS Buffer	Reagent	None	None	Prepare as described by Miltenyi.
CD4+ T cell Isolation Kit: Untouched Isolation	Reagent	Miltenyi	130-090-860	For use with the Miltenyi system.
Sterile Phosphate-Buffered Saline (PBS)	Reagent			For use in the preparation of MACS buffer.
Corning 35mm Not TC-Treated Culture Dish	Other	Corning Cell Culture Surfaces	430588	Used in the preparation of a single cell suspension.
70 micron Cell Strainer	Tool	BD Falcon	352350	Used in the preparation of a single cell suspension.
6 mL Syringe	Tool	Tyco Healthcare	8881516051	Used in the preparation of a single cell suspension.
10x Dulbecco's Phosphate Buffered Saline (DPBS)	Reagent	Invitrogen	14200-075	For use in RBC lysis.
Sterile Water	Reagent	Sigma	W4502	For use in RBC lysis.