Mouse CD4+/CD62L+/CD44 low Naive Column, Pack of 4

Assay Procedure

Kit Contents

The Mouse CD4⁺/CD62L⁺/CD44^low Naïve T Cell Enrichment Column Kit (Catalog #MCD45) contains the following components to allow for the isolation of mouse CD4⁺/CD62L⁺/CD44^low naïve T cells.

• Mouse CD4⁺/CD62L⁺/CD44^low Naïve T Cell Subset Columns
• Monoclonal Antibody Cocktail
• Column Buffer Concentrate (10X)

Other Supplies Required

Technical notes

• In order to best determine column performance, we advise that users retain a small portion of the starting cell population. Following cell selection with the column, it is then possible to perform immunophenotyping analysis on both starting and eluted cells. This information, when combined with the actual number of cells loaded and recovered, can then be used to calculate the percentage recovery of the target cell population.
• Care should be taken during cell preparation to reduce the number of platelets before loading the cells onto the column. Platelet contamination can be reduced by performing a final wash centrifugation of the cells at a reduced speed (150 - 200 x g).
• Some of the salts in the 10X column buffer solution may precipitate after storage at 4 ^°C. Should this be the case, do not carry out the 1:10 buffer dilution until all salts are in solution. This may be achieved by warming the 10X column buffer bottle in a 37 ^°C water bath for 5 - 10 minutes. Once there is no longer evidence of precipitates, the 10X column buffer may be diluted 1:10 to prepare the 1X column buffer for column processing.

Helpful Hints in Running T Cell Enrichment Columns

• Remove as many clumps as possible from the cell suspension before loading cells onto the column. Although the column is designed to filter out larger clumps of cells, too many clumps on the filter will reduce the column flow rate and cell recovery. In addition, leaving a large number of cells in a small volume of buffer for more than 30 minutes may promote cell clumping.
• If cells do not move into column after 15 minutes, the filter may have become clogged. If this occurs, move the white filter at the top of the column to the side with a sterile pipette. The cells should migrate into the column more easily.
• The column is designed so that the white filter at the top of the column bed will stop buffer flow and prevent the column from drying out. However leaving the open column exposed to air for more than 1 hour may cause the column bed to dry out and the CD4⁺ T cell enrichment to be compromised.
• Cell recovery after column processing is largely dependent on the total number of cells loaded. Optimal column performance is achieved by loading approximately 75 x10⁶ cells. Loading fewer cells will negatively affect total cell recovery.
• If buffer does not drip out of column after initial removal of the bottom cap, try tapping the side of the column to remove any air locks that may be preventing the flow of buffer.
• The white filter at the top of the column bed may be found floating as a result of being dislodged during shipping. The column can still be used for cell processing after pushing the white filter onto the column bed with the back end of a sterile 2 mL pipette. The white filter should be positioned close to the column bed.

Procedure Overview

R&D Systems Protocol for Mouse CD4⁺/CD62L⁺/CD44^low Naïve T Cell Enrichment Column

Prepare a single cell suspension of mononuclear cells.

Wash the cells with excess sterile PBS.

Remove the red blood cells if necessary (e.g., using R&D Systems Mouse Erythrocyte Lysing Kit Catalog # WL2000)

Resuspend the cells in a small volume of 1X Column Wash Buffer.

Perform a cell count.

Adjust the cell concentration to 1-2 x 10⁸ cells/mL with 1X Column Wash Buffer.

Mix 2 x 10⁸ suspended cells with the contents of one vial of Monoclonal Antibody Cocktail.

Incubate for 15 minutes at room temperature.

Wash cells twice with 10 mL 1X Column Wash Buffer.

Resuspend cells in 2 mL of 1X Column Wash Buffer.

Load cell suspension onto the prepared column.

Incubate for 10 minutes at room temperature.

Elute CD4⁺/CD62L⁺/CD44^low naïve T cells with 12 mL of 1X Column Wash Buffer.

Collect the cells in a sterile 50 mL centrifuge tube.

Pellet the cells using centrifugation.

Resuspend CD4⁺/CD62L⁺/CD44^low naïve T cells in the appropriate buffer or culture medium for downstream applications.

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