PE buffer for isolation of mononuclear cells

Prepare a solution with phosphate buffered saline (PBS), pH 7.2, and 2 mM EDTA. Keep buffer cold (2–8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).

PBE buffer for cell depletion and flow cytometry

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 1:20 with autoMACS® Rinsing Solution. Keep buffer cold (2−8 °C) and degas before using.
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as respective serum albumin, respective serum, or fetal bovine serum (FBS). Buffers or media containing Ca²⁺ or Mg²⁺ are not recommended for use.

Isolate mononuclear cells by density gradient centrifugation using Ficoll-Paque™. Follow the Ficoll-Paque protocol from the data sheet below.

Download data sheet

Isolation of mononuclear cells from bone marrow aspirates

Deplete cells expressing lineage markers from total bone marrow mononuclear cells using either the Direct Lineage Cell Depletion Kit, mouse or the Lineage Cell Depletion Kit, mouse. The first generates a high cell yield. The latter enables a more stringent depletion. Follow the protocol of the corresponding kit data sheet.

▲ Note: Lineage depleted cells can be further enriched, e.g., to obtain LSK cells, using fluorescence-activated cell sorting.

▲ Note: We recommend filtering the magnetically labeled cells before separation to guarantee a single-cell suspension either manually or via the automated protocol using the autoMACS® Pro Cell Separator. 

Download kit data sheet

Lineage Cell Depletion Kit, mouse

Direct Lineage Cell Depletion Kit, mouse

Transduce the lineage marker-negative cells with a retroviral or lentiviral vector, either directly after MACS® enrichment or after pre-stimulation culture in appropriate cell culture medium supplemented with cytokines such as SCF, TPO, FLT-3L and IL-3 and IL-6.

Use Vectofusion-1®, a novel transduction enhancer, to boost transduction efficiency. Follow the protocol of the data sheet.
 

Download data sheet

Vectofusion-1

Lineage marker-negative cells can be expanded in appropriate mouse HSC expansion media supplemented with cytokines such as SCF, TPO, FLT-3L and IL-3 and IL-6 according to standard cell culture protocols.

Lineage marker-negative cells can be further analyzed or sorted for HSC-specific surface markers by flow cytometry using MACS® Antibodies or recombinant engineered REAfinity™ antibodies.  

Use the MACS Flow Cytometry – Multicolor Panel Builder to quickly assemble the antibodies for your analysis.

The following is a list of relevant resources that might be of interest:

• StemMACS™ HSC Expansion Media XF – A xeno-free culture system for hematopoietic stem cells
• CD34 MicroBead Kit UltraPure, human – Leading the way in magnetic HSC separation
• REAfinity™ Recombinant Antibodies – Flow cytometry is in their genes
  
• REAfinity Recombinant Antibodies – Frequently Asked Questions
• Nölle, V. et al., (2016) Recombinant antibodies for improved standardization in flow cytometry.
• Watts, M.J. et al., (2008) Performance of commercial methylcellulose media for short-term colony assays in routine transplantation practice. ISCT 2008. Miami, Florida.
• StemMACS™ HSC-CFU Media – Need to estimate HSC differentiation potential? We have the media!
  

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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