PE buffer: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
▲Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).

Note:

• The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g., heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)).
  

1. Dilute cells with 2–4× the volume of PE buffer.
   ▲ Note: The more diluted the blood sample, the better the purity of the mononuclear cells.
2. Carefully layer 35 mL of diluted cell suspension over 15 mL of Ficoll-Paque™ in a 50 mL conical tube.
3. Centrifuge at 400×g for 30–40 minutes at 20 °C in a swinging-bucket rotor without brake.
4. Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.
5. Carefully transfer the mononuclear cell layer to a new 50 mL conical tube.
6. Fill the conical tube with PE buffer, mix, and centrifuge at 300×g for 10 minutes at 20 °C. Carefully remove supernatant completely.
7. For removal of platelets, resuspend the cell pellet in 50 mL of PE buffer and centrifuge at 200×g for 10–15 minutes at 20 °C. Carefully remove the supernatant completely.
   ▲ Note: This step will increase the purity of the target cells in the subsequent MACS® Cell Separation.
8. Repeat step 7. Most of the platelets will remain in the supernatant upon centrifugation at 200×g.
9. Resuspend cell pellet in an appropriate amount of PE buffer and proceed to "Isolation of NK cells".
   ▲ Note: PBMCs may be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous serum. Do not store cells longer than one day in the refrigerator. Wash at least once before proceeding to magnetic labeling and resuspend cells in an appropriate buffer. For details see MACS Cell Separation Reagents data sheets.  

Isolate pure and fully functional NK cells using the NK Cell Isolation Kit, human. This indirect magnetic labeling system isolates untouched NK cells from human PBMCs by magnetically labeling  and depleting non-NK cells (i.e., T cells, B cells, stem cells, dendritic cells, monocytes, granulocytes, and erythroid cells) with a cocktail of biotin-conjugated antibodies and the NK Cell MicroBead Cocktail. Follow the protocol in the kit data sheet.

Download the data sheet

NK Cell Isolation Kit, human

PBE Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Degas buffer before use, as air bubbles could block the column.

Notes:

• Volumes given in the data sheet for each antibody are for up to 1×10⁶ or 1×10⁷ nucleated cells. Follow the instructions provided.
  

1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Discard supernatant.
3. Resuspend according to the data sheets of the antibodies used.
4. Add antibodies according to the dilution listed in the antibody data sheets.
5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
6. Wash cells by adding 1−2 mL of PBE buffer per 1×10⁶ cells and centrifuge at 300×g for 5 minutes at 4 °C. Discard supernatant.
7. Proceed immediately to "Flow cytometry analysis".

Notes:

•  Perform the purity analysis with a cell sample taken before cell separation and a cell sample taken after isolation.
• When working with PBMCs as starting material, analyze only the original sample. 
  

After staining the samples, perform the flow cytometry analysis as follows:

1. Identify lymphocytes by SSC/CD45 gating.
2. Exclude dead cells from the analysis using the live/dead cell exclusion marker 7AAD
3. Use the SSC/FSC gate to identify different populations:
   

Ex vivo cultivation is an attractive option to increase NK cell numbers for functional analyses, including anti-tumor potential. Miltenyi Biotec NK MACS® Medium addresses the major challenges of culturing NK cells with a uniquely designed formulation that expands fully functional NK cells from either PBMCs or isolated NK cells. The medium is xeno-free and enables superior NK cell expansion with minimal growth of unwanted cells, like B, T, or DC cells.
▲Note: supplementation with serum or autologous plasma is necessary.

Complete and expansion NK MACS Medium

1. Before NK MACS Medium can be used, NK MACS Basal Medium and NK MACS Supplement (provided) must be mixed.
2. Thaw NK MACS Supplement at 2–8 °C.
3. Add 1% of NK MACS Supplement to the NK MACS Basal Medium, e.g., add 1 mL of supplement to 100 mL of basal medium.
4. Add 5% AB serum. Mix well. This is complete medium.
   ▲ Note: Only prepare the amount of complete medium which is used within 3 weeks. Complete medium can be used up to 3 weeks when stored at 2–8 °C. Do not freeze.
5. Prior to use for expansion, add 500 IU/mL of IL-2 or a combination of IL-2 and IL-15 (500 IU/mL of IL-2 and 140 IU/mL of IL-15). This is expansion NK MACS medium.
    

1. Determine the total number of cells.
2. Wash cells by adding 5–10 volumes complete medium (see "Things to prepare in advance") to the cells and centrifuge at 300×g for 10 minutes. Discard supernatant. 
3. Resuspend cells at a concentration of 1×10⁶ cells/mL in expansion NK MACS medium (see "Things to prepare in advance").
4. Pipet 300 µL of cell suspension into each well of a 24 well-plate (roughly 3×10⁵ cells/well).
   ▲  Recommended: use residual volume for cell count determination.
   

Notes:

• IMPORTANT: during NK cell cultivation, do not disturb the cells for the first week at all. After 5 days, do not change the medium. Instead, add fresh expansion NK MACS Medium (NK MACS Basal + supplement, 500 IU/mL IL-2, or a combination of 500 IU/mL IL-2 and 140 IU/mL IL-15, 5% AB serum).)
• Sampling or changing a flask: NK cells attach to the bottom of a plate or flask. Carefully disturb the cells with a 1000 µL tip pipette and resuspend cells at least 5 times. Always handle cells gently. After 10 days, aggregates may appear. This is not unusual.
• NK cell expansion is donor-dependent. The estimated volumes listed below are an example and may vary. NK cells can also be expanded for longer than 14 days, though success is also donor-dependent.

When adding expansion NK MACS medium to culture plates or flasks, please note:

• Upon reaching a well volume of 1 mL, switch to a 12-well plate (around day 5 or 7)
• Upon reaching a well volume of 2.5 mL, switch to a 6-well plate (this step can also be skipped and cells transferred directly to a T25 flask)
• Upon reaching a flask volume of 5–6 mL, switch to a T25 flask (usually around day 10 or 12)
• Upon reaching a flask volume of 7 mL, switch to a T75 flask (usually around day 12 or 14)
• Upon reaching a flask volume of 30 mL, switch to a T175 flask (usually around day 14–17)

Day 1–5

Do not disturb cells.

Day 5

Add 300µL Expansion NK MACS Medium.

Day 7

1. Take a 50 µL sample of cells to determine cell count (w/o stain, just PI/7-AAD + Scatter).
2. If the cell density is less than 1×10⁶ cells/mL, double the culture volume with fresh expansion NK MACS medium (in the case illustrated by this protocol, add 250 µL).
3. If the cell density is greater than 1×10⁶ cells/mL, add enough fresh expansion NK MACS medium to dilute cells to a final concentration of 4–5×10⁵ cells/mL (e.g., if cell density on day 7 is 1.5×10⁶ cells/mL, add 500 µL of fresh medium to the existing culture volume of 250 µL. Total volume will then be 750 µL and final cell density 5×10⁵ cells/mL).
    

Day 10 

1. Take a 50 µL sample of cells to determine cell count (w/o stain, just PI/7-AAD + Scatter).
2. Add enough fresh expansion NK MACS medium to achieve a final cell concentration of 4–5×10⁵ cells/mL.
    

Day 12 

1. Take a 50 µL sample of cells to determine cell count (w/o stain, just PI/7-AAD + Scatter).
2. Add enough fresh expansion NK MACS medium to achieve a final cell concentration of 4–5×10⁵ cells/mL.
    

Day 14

Take a 50 µL sample of cells to determine NK cell count and perform cell analysis (e.g., proceed to "Cytotoxicity assay")

 

NK cells cultivated in NK MACS® Medium retain their natural cytotoxicity against the K-562 cell line and are suitable for any downstream applications, including cytotoxicity assays and flow cytometry analysis. 

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.

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