Starting material

Human samples

Source: PBMCs from fresh whole blood or leukopak

Preparation:

• Isolate PBMCs using Ficoll‑Paque density gradient
• Wash 2× in PBS + 2% FBS (or BSA) + 1 mM EDTA
• Filter through 40 µm mesh

Typical cell composition:

T cells: 60–70% of PBMCs

B cells: 5–15% of PBMCs

Mouse samples

Source: Spleen, Lymph nodes LN, blood, or any other tissue

Preparation:

• Mechanical dissociation: spleen and LN
• Enzymatic digestion: colon, adipose or other complex tissues
• RBC lysis using ACK filter
• Wash in PBS + 2% FBS (or BSA) + 1 mM EDTA
• Filter through 40 µm mesh

Note: Adding 1 mM EDTA will reduce cell clumping.

Method 1: Negative selection

Isolation of untouched T or B cells by removing unwanted populations.

For T cell isolation:

Suggested cocktail depletion marker:

• B cells: CD19 (human), B220 (mouse)
• NK cells: CD16/CD56 (human), NK1.1 (C57BL/6 mouse) or NKp46 (other strains)
• Monocytes/macrophages: CD14/CD11b
• Dendritic cells: CD11c
• Granulocytes: Ly6G (mouse)
• Erythroid cells: Ter119 (mouse)

For B cell isolation:

Suggested cocktail depletion marker:

• T cells: CD3
• NK cells: CD16/CD56 (human), NK1.1 / NKp46 (mouse)
• Monocytes: CD14/CD11b
• Dendritic cells: CD11c
• Plasma cells (optional): CD138

Protocol

• Incubate single cell suspension with biotin-conjugated antibody cocktail
• Choose separation method:

Option A: Magnetic negative selection

• Add anti‑biotin microbeads or streptavidin‑magnetic beads to the previous mix
• Deplete labelled cells using:

Magnetic column or Static magnet: Flow‑through = untouched T or B cells

Option B: Microbubble‑based negative selection

• Add streptavidin‑coated microbubbles to the previous mix
• Separate by centrifugation

Supernatant: microbubble‑bound unwanted cells

Pellet: purified untouched B or T cells

Rationale:

Delivers untouched cells suitable for:

• Activation assays
• Signaling studies
• Transcriptomics / single‑cell workflows
• Minimizes receptor cross‑linking and activation bias

Explore our kits featuring the microbubble-based negative selection method

Method 2: Positive selection

Direct isolation of T or B cells using lineage‑specific markers.

Marker suggestions:

T cells: CD3

B cells: CD19 (human) / B220 (mouse)

Protocol

• Incubate single cell suspension with biotin‑conjugated antibody
• Add anti‑biotin microbeads / streptavidin‑magnetic beads
• Apply magnetic field with column or static magnet

Flow‑through/wash: depleted unwanted cells

Elution: highly enriched target cells

Rationale:

Best when target cells are rare or low frequency

Factors to keep in mind:  Can partially activate cells (especially column‑based selection) and if planning post‑sort labeling of CD3/CD19/B220, use different antibody clones to avoid epitope blocking.

Method 3: FACS‑based isolation

High-precision isolation of total or subset-specific T and B cells

Marker suggestions:

For T cells

• CD3
• Live/dead marker
• CD45
• Optional subset markers (eg CD4, CD8, CD62L, CD44)

For B cells

• CD19 or B220
• Live/dead marker
• CD45
• Optional subset markers (eg IgD, IgM, CD21, CD23)

Protocol

• Stain sample with chosen antibody panel and live/dead marker
• Set up the gating strategy and sort settings
• Sort target population into collection buffer
• Perform post‑sort purity QC

Rationale:

Use with a very low starting frequency or for specific subsets (eg naïve, memory, germinal center) or if highest purity is required (99%).

Factors to keep in mind: longer processing time, lower yield, higher cellular stress.