Streptavidin Nanobeads（300 nm）

Steps

Operating instructions

Dosage and timing

1

Collect the mouse spleen, grind it on a 40 μm sterile filter mesh to prepare a single-cell suspension of splenic cells; after red blood cell lysis, proceed with cell counting;

Note:It is recommended to count cells after staining with AO/PI; splenic cells can be centrifuged at 400 g for 7 minutes;

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 for initial Mouse T cell ratio testing.

1 × 10⁷cells/100 μL

3

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

Note:For this step, gentle pipetting up and down 2–3 times can be used to mix.

10 μL / 100 μL

4

Incubate the antibodies in the cocktail with the cells at room temperature for 10 minutes.

Incubate at room temperature for 10 minutes.

5

Shake for 5–30 seconds to mix the Streptavidin Nanobeads (300 nm).

Mix for 5–30 seconds.

6

Add 10 μL of Streptavidin Nanobeads (300 nm) to the sample.

10 μL per 100 μL.

7

Gently mix the magnetic beads and cells, and incubate at room temperature for 5 minutes.

Note:In this step, the mixture can be gently pipetted up and down 2–3 times to homogenize;

During this step, the magnetic beads may settle at the bottom. You can gently tap the tube to mix them after 2.5 minutes;

Incubate at room temperature for 5 minutes

8

Add MagSep Separation Buffer to the sample and immediately place it on the magnetic stand to prevent the magnetic beads from settling excessively;

Adjust the total volume to 2.5 mL (in a 5 mL flow cytometry tube)

Adjust the total volume to 7.5 mL (in a 15 mL centrifuge tube)

9

Place the sample on the magnetic stand to allow the magnetic beads to adhere to the tube wall;

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

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

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 at 400g for 7 minutes for subsequent culture and analysis.

Cell sorting successful

Note: 1.0.1-1 × 10^8 cells (0.1-1 mL): it is recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) to capture a total volume of 2.5 mL;

2-5 × 10^8 cells (2-5 mL): it is recommended to use a 15 mL multi-well magnetic separator (Starter EasyEights EasyIso Separator) to capture a total volume of 7.5 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);