Mouse CD4⁺ Cell Isolation Kit (Positive Isolation) (C008B-270033)

The Mouse CD4⁺ Cell Isolation Kit (Positive Isolation) is used to isolate CD4⁺ cells from mouse spleen or other tissue single-cell suspensions by positive i solation. The principle is to label CD4⁺ cells with a CD4 Capture Antibody, then capture the target cells using Releasable Magnetic Beads, and finally dissociate the beads from the cell surface using a Magnetic Beads Release Buffer, thereby obtaining magnetic bead‑free mouse CD4⁺ cells. The isolated CD4⁺ cells can be used in downstream molecular and cell biology experiments.ApplicationsIsolation of CD4⁺ cells from mouse lymphoid organs such as spleen and lymph nodes.M1522421ComponentAppearance10T100TStorageM1522421ACD4 Capture AntibodyLiquid20 μL200 μL2-8℃.M1522421BReleasable Magnetic BeadsLiquid200 μL1 mL ×22-8℃.M1522421CMagnetic Beads Release BufferLiquid4 mL40 mL2-8℃.Protocol (Using Mouse Spleen CD4⁺ 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: A small amount of residual red blood cells 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 filtered 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 CD4 Capture AntibodyAdd 500 μL of cell suspension (5×10⁷ cells) to the bottom of a sterile FACS tube, then add 10 μL of CD4 Capture Antibody. Mix well and incubate at 4°C for 15 min.Note: Add the cell suspension directly 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. For other cell numbers, adjust the volume of CD4 Capture Antibody proportionally. If isolating fewer than 1×10⁷ cells, adjust the cell suspension volume to 100 μL and add 2 μL of CD4 Capture Antibody.Add Releasable Magnetic BeadsAfter incubation, add 100 μL of washed Releasable Magnetic Beads to the FACS tube, mix well, and incubate at 4°C for 15 min (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).Note: For other cell numbers, adjust the volume of Releasable Magnetic Beads proportionally. If isolating fewer than 1×10⁷ cells, use 20 μL of Releasable Magnetic Beads.First magnetic separation and washAfter incubation, add Isolation Buffer to the FACS tube to a total volume of 2.5 mL, and mix by pipetting 5 times (avoid inverting the tube). Place the FACS tube on a magnetic separator and let stand for 5 min.Discard supernatant and resuspend beadsAspirate and discard the supernatant. Remove the tube from the magnetic separator, quickly add 2 mL of Isolation Buffer, and pipette repeatedly to disperse the beads. Place the tube back on the magnetic separator and let stand for 5 min.Repeat washingRepeat step 8 twice (thorough washing ensures high purity of target cells after subsequent release).First elutionAfter magnetic separation, aspirate and discard the supernatant. Remove the tube from the magnetic separator, quickly add 1 mL of Magnetic Beads Release Buffer to resuspend the beads (avoid letting the beads dry), transfer the bead suspension to a 1.5 mL centrifuge tube, and rotate at room temperature for 10 min.Note: For other cell numbers, adjust the volume of Magnetic Beads Release Buffer proportionally. If isolating fewer than 1×10⁷ cells, use 200 μL of Magnetic Beads Release Buffer for elution.Collect first elution cellsAfter incubation, pipette repeatedly at least 10 times, transfer the bead suspension to a new FACS tube, add Isolation Buffer to 2.5 mL, mix by pipetting, place the tube on a magnetic separator, and let stand for 5 min.Second elutionTransfer the supernatant to a 15 mL centrifuge tube and keep it (the supernatant contains the target cells; do not discard). Quickly resuspend the beads in 1 mL of Magnetic Beads Release Buffer (avoid drying), transfer to a 1.5 mL centrifuge tube, and rotate at room temperature for 10 min.Collect second elution cellsAfter incubation, pipette repeatedly at least 10 times, transfer the bead suspension to a new FACS tube, add Isolation Buffer to 2.5 mL, mix by pipetting, place the tube on a magnetic separator, and let stand for 5 min.Combine cells and centrifugeCombine the supernatant with the cell supernatant from the first elution, centrifuge at 500 g for 5 min, discard the supernatant, and collect the magnetic bead‑free CD4⁺ 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⁺ cells were isolated from BALB/c mouse splenocytes. Cells before and after isolation were stained with PE‑labeled anti‑mouse CD4 antibody (clone RM4‑4) and analyzed by flow cytometry. The purity of CD4⁺ cells before and after isolation was 13.3% and 97.2%, respectively.PrecautionsAvoid freezing any component of the kit 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⁺ Cell Isolation Kit (Positive Isolation) is used to isolate CD4⁺ cells from mouse spleen or other tissue single-cell suspensions by positive i solation. The principle is to label CD4⁺ cells with a CD4 Capture Antibody, then capture the target cells using Releasable Magnetic Beads, and finally dissociate the beads from the cell surface using a Magnetic Beads Release Buffer, thereby obtaining magnetic bead‑free mouse CD4⁺ cells. The isolated CD4⁺ cells can be used in downstream molecular and cell biology experiments.ApplicationsIsolation of CD4⁺ cells from mouse lymphoid organs such as spleen and lymph nodes.M1522421ComponentAppearance10T100TStorageM1522421ACD4 Capture AntibodyLiquid20 μL200 μL2-8℃.M1522421BReleasable Magnetic BeadsLiquid200 μL1 mL ×22-8℃.M1522421CMagnetic Beads Release BufferLiquid4 mL40 mL2-8℃.Protocol (Using Mouse Spleen CD4⁺ 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: A small amount of residual red blood cells 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 filtered 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 CD4 Capture AntibodyAdd 500 μL of cell suspension (5×10⁷ cells) to the bottom of a sterile FACS tube, then add 10 μL of CD4 Capture Antibody. Mix well and incubate at 4°C for 15 min.Note: Add the cell suspension directly 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. For other cell numbers, adjust the volume of CD4 Capture Antibody proportionally. If isolating fewer than 1×10⁷ cells, adjust the cell suspension volume to 100 μL and add 2 μL of CD4 Capture Antibody.Add Releasable Magnetic BeadsAfter incubation, add 100 μL of washed Releasable Magnetic Beads to the FACS tube, mix well, and incubate at 4°C for 15 min (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).Note: For other cell numbers, adjust the volume of Releasable Magnetic Beads proportionally. If isolating fewer than 1×10⁷ cells, use 20 μL of Releasable Magnetic Beads.First magnetic separation and washAfter incubation, add Isolation Buffer to the FACS tube to a total volume of 2.5 mL, and mix by pipetting 5 times (avoid inverting the tube). Place the FACS tube on a magnetic separator and let stand for 5 min.Discard supernatant and resuspend beadsAspirate and discard the supernatant. Remove the tube from the magnetic separator, quickly add 2 mL of Isolation Buffer, and pipette repeatedly to disperse the beads. Place the tube back on the magnetic separator and let stand for 5 min.Repeat washingRepeat step 8 twice (thorough washing ensures high purity of target cells after subsequent release).First elutionAfter magnetic separation, aspirate and discard the supernatant. Remove the tube from the magnetic separator, quickly add 1 mL of Magnetic Beads Release Buffer to resuspend the beads (avoid letting the beads dry), transfer the bead suspension to a 1.5 mL centrifuge tube, and rotate at room temperature for 10 min.Note: For other cell numbers, adjust the volume of Magnetic Beads Release Buffer proportionally. If isolating fewer than 1×10⁷ cells, use 200 μL of Magnetic Beads Release Buffer for elution.Collect first elution cellsAfter incubation, pipette repeatedly at least 10 times, transfer the bead suspension to a new FACS tube, add Isolation Buffer to 2.5 mL, mix by pipetting, place the tube on a magnetic separator, and let stand for 5 min.Second elutionTransfer the supernatant to a 15 mL centrifuge tube and keep it (the supernatant contains the target cells; do not discard). Quickly resuspend the beads in 1 mL of Magnetic Beads Release Buffer (avoid drying), transfer to a 1.5 mL centrifuge tube, and rotate at room temperature for 10 min.Collect second elution cellsAfter incubation, pipette repeatedly at least 10 times, transfer the bead suspension to a new FACS tube, add Isolation Buffer to 2.5 mL, mix by pipetting, place the tube on a magnetic separator, and let stand for 5 min.Combine cells and centrifugeCombine the supernatant with the cell supernatant from the first elution, centrifuge at 500 g for 5 min, discard the supernatant, and collect the magnetic bead‑free CD4⁺ 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⁺ cells were isolated from BALB/c mouse splenocytes. Cells before and after isolation were stained with PE‑labeled anti‑mouse CD4 antibody (clone RM4‑4) and analyzed by flow cytometry. The purity of CD4⁺ cells before and after isolation was 13.3% and 97.2%, respectively.PrecautionsAvoid freezing any component of the kit 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:

7.06 Ounces

HazmatClass:

No

Quantity:

1

Unit of Measurement:

ST

WeightUOM:

LB

MPN:

M1522421-10T

Country of Origin:

China

Product Size:

10T

Harmonized Tariff Code:

2931.90.90