Mouse Neutrophil Isolation Kit (Column-Free)

Steps

Operating Instructions

Dosage and Time

1

Collect mouse bone marrowor peripheral bloodand prepare a single-cell suspension,Perform cell counting;

Note:Mouse peripheral blood requiresthe use ofred blood cell lysis bufferRed Blood Cell Lysis Buffer (without fixative, 1X)(cat:S0D9002) to perform red blood cell lysis;; It is recommended to stain cells withAO/PIbefore counting;;

Prepare the sample

2

Use100 μLMagSep Separation Bufferto resuspend1×10⁷cells;

Note:Reagents should be adjusted proportionally according to the sample volume; set aside a pre-sorting sample to determine the initialMouse Neutrophilratio;

1×10⁷cells/100 μL

3

Add10 μL MouseNeutrophilBiotin Antibody Cocktailto the sample, and gently mix;

Note:For this step, gentle mixing can be achieved by pipetting up and down.2-3below;

10 μL / 100 μL

4

Incubate the antibodies in the Cocktail with the cells.,2-8℃incubation10 min;2

-8℃ incubation10 min5

5

Vortexfor 5–30 secondsto mix wellStreptavidin beads;mix for 5–30 seconds

6add

6

to the sample;15 μL/100 μL7

gently mix the magnetic beads and cells,

2

-8℃incubate15min5minutes;

Note:In this step, gentle mixing can be achieved by gently pipetting up and down2-3times;

In this step, the magnetic beads may settle at the bottom; you can gently tap the tube twice to remix them;

2-8℃incubation15minutes;

8

addMagSep Separation BufferAdd to the sample and immediately place it on the magnetic pole to prevent the magnetic beads from settling at the bottom in large quantities;

Increase the total volume to2.5 mL(5 mLflow cytometry tube)

Increase the total volume to7.5 mL(15 mLcentrifuge tube)

9

Place the sample on the magnetic pole so that the magnetic beads adhere to the tube wall;

Adsorb using a single-well magnetic rack (3min)

Adsorb using a multi-well magnetic rack (5min)

10

Tilt the magnetic rack and pour the sample into a new collection tube to collect the sample;

Note:The sorted cells can be centrifuged at400gfor7 min, for subsequent culture and analysis.

Cell sorting successful

Note:1. 0.1-1 × 10⁸cells(0.1-1mL) are recommended to use5 mLsingle-hole magnetic pole(Starter EasyIso Separator)the total adsorption volume is2.5mL;

2-5 ×10⁸cells(2-5mL) are recommended to use15 mLTandem magnetic poles(Starter EasyEights EasyIso Separator)The total adsorption volume is7.5 mL;

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);