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Identification Protocol of Mouse Regulatory T cell

Source: Elabscience®Published: Jan 18,2024

Identification Procedure of Mouse Regulatory T cell


Experimental Design

Cell subpopulation
Species
Marker
Fluorescence labeling
Clone number
Treg
Mouse
CD4
FITC
GK1.5
CD25
APC
PC-61.5.3
Foxp3
PE
3G3


Flow Cytometry Antibodies Required for Mouse Regulatory T cell Identification

Marker
Fluorescence
Cat. No.
Specification
CD4
FITC
100T
CD25
APC
20T
Foxp3
PE
20T


Auxiliary Reagents Required for Mouse Regulatory T cell Identification

Purpose
Cat. No.
Product Name
Specification
Isotype control
APC Rat IgG1, κ Isotype Control
20T
Isotype control
PE Mouse IgG1, κ Isotype Control
20T
Cell staining buffer
Cell Staining Buffer
100 mL
Red blood cell lysate
10×ACK Lysis Buffer
100 mL
Fixation and Permeabilization buffer
Foxp3/Transcription Factor Staining Kit
20 Assays


Panel Design

Purpose
Sample
Antibody Collocation
Adjust the voltage
1
Black
Adjust compensation
2
CD4-FITC
3
CD25-APC
4
Foxp3-PE
APC-FMO in combination with Isotype Control for auxiliary gating
5
CD4-FITC, Foxp3-PE; Rat IgG1, κ Isotype Control- APC
PE-FMO in combination with Isotype Control for auxiliary gating
6
CD4-FITC, CD25-APC; Mouse IgG1, κ Isotype Control- PE
Full Panel
7
CD4-FITC, CD25-APC, Foxp3-PE


Experimental Procedure

Steps for isolating mouse spleen cells:

1)Take mouse spleen and place it in PBS.

2)Grind the spleen with a syringe plunger, use 70 μM cell filter for filtration and collect the filtrate into a 15 mL centrifuge tube.

3)Centrifuge at 300 ×g for 5 min, and obtain red cell precipitate.

4)Add 2 mL of 1× erythrocyte lysate to resuspend the cells. After lysing at room temperature for 2~3 minutes, immediately add 10 mL of PBS to terminate the lysis reaction.

5)Centrifuge the solution at 300 g for 5 minutes, discard the supernatant, and obtain red cell precipitate. Resuspend the spleen cells with cell staining buffer and perform cell counting, adjusting cell concentration to 1×107/mL.


Steps of Fixation and Permeabilization:

(The following are the experimental steps for the full staining tube. The Blank tube does not add antibodies, while the Single staining tube only adds corresponding Flow Cytometry antibodies in the corresponding steps. The Isotype control tube replaces the corresponding Flow Cytometry antibodies with the Isotype control antibody, and all tubes need to undergo Fixation and Permeabilization.)

1)Dilute Foxp3 detection specific Fixation and Permeabilization buffer based on the amount detected.

2)Take 100 μL mouse spleen cells1×106 cells) into an EP tube. Add 1 Test FITC Anti-Mouse CD4 (E-AB-F1097C) and 1Test APC Anti-Mouse CD25 (E-AB-F1102E), mixed. Incubated at 4 ℃ for 30 minutes.

3)After incubation, add 1 mL of Cell Staining Buffer, centrifuge at 300 × g for 5 minutes. Discard the supernatant and add 100 μL of Cell Staining Buffer to resuspend cells.

4)Add 1 mL of Fixation Working Solution, vortex mix well and react at 4℃ for 30 minutes. After the reaction is completed, centrifuge at 600 × g for 5 minutes and discard the supernatant.

5)Add 2 mL of Permeabilization Working Solution, vortex mix well and react at 4℃ for 30 minutes. After the reaction is completed, centrifuge at 600 × g for 5 minutes and discard the supernatant.

6)Repeat Step 5.

7)Add 100 μL of 1× Permeabilization Working Solution to resuspend the sample. Then add PE Anti-Mouse Foxp3 (E-AB-F1238D), mix well and react at RT for 30 minutes.

8)Add 2 mL of Permeabilization Working Solution, centrifuge at 600 × g for 5 minutes and discard the supernatant.

9)Add 200 μL cell staining buffer (E-CK-A107) to resuspend the sample. Conduct the detection.


Experimental Result

Detection of Treg (3-color) in Mouse Spleen

Tips:

▶ There is fluorescence spillover, so it is necessary to set single positive tubes for compensation.

▶ CD4 cell population is obvious, and there is no need of Isotype Control. But CD25 and Foxp3 populations are not obvious, and Isotype Controls are needed.

▶ This experiment was validated using normal mouse spleen without any biological control.

▶ Gating:

1)Gating the target cells based on FSC-A and SSC-A.

2)Utilizing the light scattering characteristics to remove adhesions and obtain single cells.

3)Determine CD4+ Th cells through CD4.

4)Identifying Treg cell subpopulations through CD25+/Foxp3+.