Mouse CD4⁺ T Cell Isolation Kit (Negative Isolation) (C008B-266881)

The Mouse CD4⁺ T Cell Isolation Kit (Negative Isolation) is used to isolate CD4⁺ T cells from mouse spleen or other tissue single-cell suspensions by negative i solation. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells (non-CD4⁺ T cells), followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the isolation of mouse CD4⁺ T cells. A magnetic separator is required for the isolation process.ApplicationsIsolation of CD4⁺ T cells from mouse spleen and lymph nodes.M1522407ComponentAppearance10T50T100TStorageM1522407ABiotin-Antibody MixLiquid20 μL100 μL200 μL2-8℃.M1522407BStreptavidin Magnetic BeadsLiquid200 μL1 mL1 mL×22-8℃.Protocol (Using Mouse Spleen CD4⁺ T Cell Isolation as an Example)Prepare single-cell suspensionGrind the spleen on a 70 μm cell strainer, rinse the strainer with pre-chilled PBS, collect the cell suspension into a 50 mL centrifuge tube, and centrifuge at 500 g for 5 min.Lyse red blood cellsAfter centrifugation, discard the supernatant, add 5 mL of ACK red blood cell lysis buffer, lyse at room temperature for 5 min, then add 20 mL PBS and centrifuge at 500 g for 5 min.Note: The red blood cell lysis step can be adjusted in volume and time depending on the lysis buffer used. A small amount of red blood cell residue will not affect subsequent isolation or cell purity.Filter and count cellsAfter centrifugation, discard the supernatant, resuspend the splenocytes in PBS, filter the cell suspension through a 70 μm cell strainer, and count the cells. After counting, centrifuge at 500 g for 5 min.Note: The cell suspension must be passed through a cell strainer to remove tissue and cell clumps; otherwise, subsequent cell isolation purity may be affected.Adjust cell densityAfter centrifugation, discard the supernatant, resuspend the cells in Isolation Buffer to a density of 1×10⁸ cells/mL.Note: Isolation buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS), or PBS containing 2 mM EDTA and 0.5% BSA. The buffer must be pre-filtered through a 0.22 μm filter.Add Biotin-Antibody MixAdd 100 μL of cell suspension (1×10⁷ cells) to the bottom of a sterile FACS tube, then add 2 μL of Biotin-Antibody Mix. Mix well and incubate at 4°C for 10 min.Note:When adding the cell suspension, add it to the bottom of the tube, avoiding the tube wall.Depending on the magnetic separator used, a centrifuge tube can also be used for cell isolation.If isolating more cells, increase the volume of Biotin-Antibody Mix proportionally.Add Streptavidin Magnetic BeadsAfter incubation, add 20 μL of washed Streptavidin Magnetic Beads to the FACS tube (the beads must be washed before use: vortex to resuspend the beads, transfer the required volume of beads to a 1.5 mL centrifuge tube, add 1 mL of Isolation Buffer, centrifuge at 10,000 g for 1 min or use a magnetic separator for 3 min, then discard the supernatant. Add 1 mL of Isolation Buffer and wash the beads once more, then resuspend the beads in the same volume of Isolation Buffer as originally taken. For example, if 20 μL of beads are taken, resuspend them in 20 μL of Isolation Buffer after washing). Mix well and incubate at 4°C for 10 min.Note:If isolating more cells, increase the volume of Streptavidin Magnetic Beads proportionally. For example, to isolate 5×10⁷ cells, add 10 μL of Biotin-Antibody Mix and 100 μL of Streptavidin Magnetic Beads to 500 μL of cell suspension.If isolating fewer than 1×10⁷ cells, adjust the cell suspension volume to 100 μL, then add 2 μL of Biotin-Antibody Mix and 20 μL of Streptavidin Magnetic Beads.Dilute and mixAfter incubation, add 2.5 mL of Isolation Buffer to the FACS tube, and mix gently by pipetting up and down 5 times (avoid vigorous shaking or inverting).Magnetic separationPlace the FACS tube containing the cell suspension on a magnetic separator and let stand for 5 min.Collect CD4⁺ T cellsGently pour the cell suspension into a sterile centrifuge tube (do not remove the FACS tube from the magnetic separator while pouring). This cell suspension contains the purified mouse CD4⁺ T cells. Centrifuge at 500 g for 5 min, then discard the supernatant and collect the cells.Resuspend for further useWash the cells as required for your experiment, then resuspend them in the desired buffer or culture medium for subsequent molecular or cell biology experiments.Isolation PerformanceCD4⁺ T cells were isolated from C57BL/6 mouse splenocytes. Cells before and after isolation were stained with FITC anti-mouse CD4 antibody (clone GK1.5) and analyzed by flow cytometry. The purity of CD4⁺ T cells before and after isolation was 20.3% and 98.8%, respectively.PrecautionsAvoid freezing the magnetic beads and antibody mix during use and storage.Low-retention pipette tips and centrifuge tubes are recommended to avoid loss of beads and antibodies due to adsorption.This product must be used with a magnetic separator.For research use only.

Specifications & Purity: The Mouse CD4⁺ T Cell Isolation Kit (Negative Isolation) is used to isolate CD4⁺ T cells from mouse spleen or other tissue single-cell suspensions by negative i solation. The principle is to use a cocktail of biotin-labeled monoclonal antibodies against non-target cells (non-CD4⁺ T cells), followed by removal of these non-target cells using streptavidin-labeled magnetic beads, thereby achieving the isolation of mouse CD4⁺ T cells. A magnetic separator is required for the isolation process.ApplicationsIsolation of CD4⁺ T cells from mouse spleen and lymph nodes.M1522407ComponentAppearance10T50T100TStorageM1522407ABiotin-Antibody MixLiquid20 μL100 μL200 μL2-8℃.M1522407BStreptavidin Magnetic BeadsLiquid200 μL1 mL1 mL×22-8℃.Protocol (Using Mouse Spleen CD4⁺ T Cell Isolation as an Example)Prepare single-cell suspensionGrind the spleen on a 70 μm cell strainer, rinse the strainer with pre-chilled PBS, collect the cell suspension into a 50 mL centrifuge tube, and centrifuge at 500 g for 5 min.Lyse red blood cellsAfter centrifugation, discard the supernatant, add 5 mL of ACK red blood cell lysis buffer, lyse at room temperature for 5 min, then add 20 mL PBS and centrifuge at 500 g for 5 min.Note: The red blood cell lysis step can be adjusted in volume and time depending on the lysis buffer used. A small amount of red blood cell residue will not affect subsequent isolation or cell purity.Filter and count cellsAfter centrifugation, discard the supernatant, resuspend the splenocytes in PBS, filter the cell suspension through a 70 μm cell strainer, and count the cells. After counting, centrifuge at 500 g for 5 min.Note: The cell suspension must be passed through a cell strainer to remove tissue and cell clumps; otherwise, subsequent cell isolation purity may be affected.Adjust cell densityAfter centrifugation, discard the supernatant, resuspend the cells in Isolation Buffer to a density of 1×10⁸ cells/mL.Note: Isolation buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS), or PBS containing 2 mM EDTA and 0.5% BSA. The buffer must be pre-filtered through a 0.22 μm filter.Add Biotin-Antibody MixAdd 100 μL of cell suspension (1×10⁷ cells) to the bottom of a sterile FACS tube, then add 2 μL of Biotin-Antibody Mix. Mix well and incubate at 4°C for 10 min.Note:When adding the cell suspension, add it to the bottom of the tube, avoiding the tube wall.Depending on the magnetic separator used, a centrifuge tube can also be used for cell isolation.If isolating more cells, increase the volume of Biotin-Antibody Mix proportionally.Add Streptavidin Magnetic BeadsAfter incubation, add 20 μL of washed Streptavidin Magnetic Beads to the FACS tube (the beads must be washed before use: vortex to resuspend the beads, transfer the required volume of beads to a 1.5 mL centrifuge tube, add 1 mL of Isolation Buffer, centrifuge at 10,000 g for 1 min or use a magnetic separator for 3 min, then discard the supernatant. Add 1 mL of Isolation Buffer and wash the beads once more, then resuspend the beads in the same volume of Isolation Buffer as originally taken. For example, if 20 μL of beads are taken, resuspend them in 20 μL of Isolation Buffer after washing). Mix well and incubate at 4°C for 10 min.Note:If isolating more cells, increase the volume of Streptavidin Magnetic Beads proportionally. For example, to isolate 5×10⁷ cells, add 10 μL of Biotin-Antibody Mix and 100 μL of Streptavidin Magnetic Beads to 500 μL of cell suspension.If isolating fewer than 1×10⁷ cells, adjust the cell suspension volume to 100 μL, then add 2 μL of Biotin-Antibody Mix and 20 μL of Streptavidin Magnetic Beads.Dilute and mixAfter incubation, add 2.5 mL of Isolation Buffer to the FACS tube, and mix gently by pipetting up and down 5 times (avoid vigorous shaking or inverting).Magnetic separationPlace the FACS tube containing the cell suspension on a magnetic separator and let stand for 5 min.Collect CD4⁺ T cellsGently pour the cell suspension into a sterile centrifuge tube (do not remove the FACS tube from the magnetic separator while pouring). This cell suspension contains the purified mouse CD4⁺ T cells. Centrifuge at 500 g for 5 min, then discard the supernatant and collect the cells.Resuspend for further useWash the cells as required for your experiment, then resuspend them in the desired buffer or culture medium for subsequent molecular or cell biology experiments.Isolation PerformanceCD4⁺ T cells were isolated from C57BL/6 mouse splenocytes. Cells before and after isolation were stained with FITC anti-mouse CD4 antibody (clone GK1.5) and analyzed by flow cytometry. The purity of CD4⁺ T cells before and after isolation was 20.3% and 98.8%, respectively.PrecautionsAvoid freezing the magnetic beads and antibody mix during use and storage.Low-retention pipette tips and centrifuge tubes are recommended to avoid loss of beads and antibodies due to adsorption.This product must be used with a magnetic separator.For research use only.

Grade: BioReagent

Storage Temperature: Store at 2-8°C;Do not freeze

Shipped in: Wet ice;Do not freeze

UPC:

41106514

Weight:

0.70 Ounces

HazmatClass:

No

Quantity:

1

Unit of Measurement:

ST

WeightUOM:

LB

MPN:

M1522407-50T

Country of Origin:

China

Product Size:

50T

Harmonized Tariff Code:

2931.90.90