Issue 2July, 2014

Cell separation (1) THP-1
Introduction
THP-1 is a human monocytic cell line. This cell differentiates into macrophage by PMA (Phorbol 12-Myristate 13-Acetate) stimulation, and is used inmacrophage studies. We performed model experiments of cell separation using THP-1.

SummaryWe evaluated the cell separation ability of commercially available Streptavidin immobilized magnetic beads using the biotin-labeled THP-1.
FG beads have a higher collection rate than other beads in our study.

Result 1 (Column)
We compared FG beads with other commercially available magnetic beads byuse of Magnetic column.
We confirmed FG beads have a superior ability for the collection of live THP-1.
This verifies FG beads are useful for the cell separation application.

Result 2 (Stand)

Beads

Collection rate (%)

Standard deviation

FG beads

35.8

3.3

A

9.7

4.6

B

6.9

4.5

C

18.7

9.7

D

5.9

3.5

*MACS Column MS and Mini MACS Separator (Miltenyi Biotec)

We compared FG beads with competitor A and B by use of Magnetic stand.
We confirmed FG beads have a superior ability for the collection of live THP-1.
Beads

Collectionrate (%)

Survival rate (%)
after 24h

Survival rate (%)

Proliferation rate (%)
after 24h

Non(untreate)

-9798

190

FG beads

61.3

9599190A

1.08691

200

B

4.8

9896140*Magnet Stand (cat#TA4899N12)

Result 3 (Flow cytometry)
We compared FG beads with competitor A and B by use of Magnetic column.
Evaluation was performed by the flow cytometry.
‘Upper column’ is the number of cells which were trapped in the column.
‘Lower column’ is the number of cells which passed through the column.

Result 4 (Methods)
AAB
FG
We compared antibody binding methods.
‘cells’ : Cells were labeled with biotinylated antibody first, then biotin-labeled cells were separated by Streptavidin beads.
‘beads’ : Biotinylated antibodies were bound to Streptavidin beads first, then cells were separated by antibody-immobilized beads.
We experienced better results with the biotinylation of cells.

AntibodyCollection
rate (%)

Survival
rate (%)

Survival rate (%)
after 24h

FG

Proliferation rate (%)
after 24h

Non(untreate)

-9597

170

cells

50.0

9497

180

beads

5.09195140

TAMAGAWA SEIKI CO., LTD.

B

*Magnet Stand (cat#TA4899N12)

http://www.FGbeads.com

Experimental Information
THP-1(20um) FG beads(0.2um)
Biotinylation of cells

Magnetic labeling

Cell separation

Streptavidinbeads

Biotinylatedantibody
AntigenMaterials and method■Materials■Methods 1 (Biotinylation of THP-1 cells)

1. Streptavidin beads(cat#TAS8848N1170 and competitor A, B, C and D)
2. THP-1 Cell line
3. Biotinylated CD14 antibody (SM3009B, Acris Antibodies)
4. Labeling Buffer (PBS(pH7.2), 2mM EDTA, 0.5% BSA)
5. Separation Buffer (PBS(pH7.2), 2mM EDTA, 0.5% BSA)
6. Cell counting Kit 8 (Dojin Chemical)
7. Trypan Blue (Bio-Rad)■Apparatus
1. Magnetic Stand : Magnet Stand (cat#TA4899N12)
2. Column : MACS Column MS (Miltenyi Biotec)
Mini MACS Separator (Miltenyi Biotec)

1. Add biotinylated antibody to THP-1 Cells
Add 500ul of 1/250 diluted antibody to 107 THP-1 cells in Labeling Buffer.
2. Reaction
Mix for 10min at 4℃.
3. Wash and Store
Remove the unreacted antibody by centrifugation.
Wash with 500ul of Labeling buffer 2 times.
Add 90 ul of Labeling buffer to the precipitated biotin-labeled THP-1 cells.

■Methods 2 (Magnetic-labeling of THP-1 cells)
1. Add beads to THP-1 cells
Add 0.1mg (10ul) of Streptavidin beads to 90ul of biotin-labeled THP-1 cells.
2. Reaction
Mix for 15min at 4℃.

■Methods 3 (Wash and separation of beads-bound cells)

Magnet Stand (cat#TA4899N12)Column1. Apply to column
Apply cell suspension to MACS Column and Separator.
2. Wash and Recover
Wash with 500ul of Separation Buffer 3 times.
If column is used, remove the cplumn from the Separator and thencollect beads-bound cells from the column with injector.
Transfer to culture medium.
Stand
Note: When Magnet Stand is used, take care not to suction cells when removing
supernatant, because cell pellet may not be completely intact aftermagnetic separation.

■Methods 4 (Evaluation)
MACS Column MS and Mini MACS Separator(Miltenyi Biotec)

TAMAGAWA SEIKI CO., LTD.

Count cell viability with Cell Counting Kit 8 (Result 1), or Trypan Blue (Result 2).

http://www.FGbeads.com