Mouse Neutrophil Isolation Kit (Column-Based)

Steps

Operating instructions

Dosage and time

Cell processing and labeling

1

Collect mouse bone marrow or peripheral blood, prepare a single-cell suspension, and perform cell counting;

Note:Mouse bone marrow and peripheral blood must undergo red blood cell lysis using Red Blood Cell Lysis Buffer (without fixative, 1X) (Cat: S0D9002); it is recommended to count cells after staining with AO/PI;

Prepare the sample

2

Resuspend 1 × 10^7 cells in 100 μL of MagSep Separation Buffer;⁷cells;

Note:Reagents should be adjusted proportionally according to the sample volume; retain a pre-sorting sample to determine the proportion of mouse neutrophils;

1×10⁷cells/100 μL

3

Add 10 μL of Mouse Neutrophil Biotin Antibody Cocktail to the sample and gently mix.

Note:For this step, gently pipette up and down 2–3 times to mix.

10 μL/100 μL

4

Incubate the antibodies in the cocktail with the cells at 2–8°C for 10 minutes.

Incubate at 2–8°C for 10 minutes

5

Mix wellStreptavidin Beads, add 15 μLStreptavidin Beadsto the sample;

15 μL/100 μL

6

Gently mix the magnetic beads and cells, and incubate at 2–8°C for 15 minutes;

Incubate at 2–8°C for 15 minutes

7

Add MagSep Separation Buffer equal to ten times the cell volume to wash the cells, centrifuge at 400g for 7 minutes, discard the supernatant, and resuspend to the original volume;

Centrifuge at 400g for 7 minutes

Cell sorting

8

In the column selection magnet, installL Separation Column, add 3 mL of MagSep Separation Buffer to rinse the separation column;

Rinse the separation column

9

Resuspend the cells in 1 mL of MagSep Separation Buffer and load them into the separation column, collecting the target cells in a collection tube;

Note:The cell volume should be at least 500 μL; if insufficient, make up the volume with MagSep Separation Buffer; the cells that drip down under gravity are the target cells;

Collect the target cells

10

Add 3 mL of MagSep Separation Buffer three times, collecting the target cells;

Note:This step elutes the unlabeled target cells from the separation column, increasing the yield.

Collect the target cells

Notes: 1. Pay attention to the maximum capacity of the separation column,The S Separation Column can hold a maximum of1×10⁷cells;The L Separation Column has a maximum capacity of1×10⁸;

2. If the sample viability is low, it is recommended to use a dead cell removal kit to eliminate dead cells (Dead Cell Removal Set, (RUO)#S0K0009);