-Detailed Information [IFO50520]-

Notes for Cultivation of MG5 Cells

[Culture Medium]

Mixture of 30% Dulbecco's modified Eagle's medium(*a) containing 10% fetal bovine serum (FBS) and 70% A1 conditioned medium(*b) (or 100% conditioned medium of astrocytes).

*a) Dulbecco's modified Eagle's medium with 4.5 g/L glucose. For example, GBCO BRL #12100-038.

*b) Preparation of A1-conditioned medium.
1. Grow A1 cells (IFO50519) in 10% FBS-DMEM (high glucose) to sub-confluency on T-150 flasks.

2. Replace the medium with 60 mL fresh 10% FBS-DMEM and culture the cells overnihgt.

3. Collect the culture medium and filtrate it using a filter unit (pore size, 0.22 um). Store the filtered medium (A1-conditioned medium) at -80 C.

4. Please take care not to contaminate the A1 cells to MG5 cells. Avoid sharing the media, enzymes, buffers, or other equipments between A1 and MG5.

[Culture Substrata]

Bacterial grade dish (such as Falcon #1001, #1016) or flask (such as Falcon #3009, #3012).

[Thawing Frozen Cells and Cultivation of MG5 cells]

1. Thaw the frozen vial quickly by vigorously shaking the vial in 37 C water bath.

2. Strilize the surface of vials with 70% ethanol. Surround the vial with sterile quaze and snap it at the neck.

3. Transfer the vial content to 10 mL culture medium in 15 mL tube, and centrifuge at 1000 rpm for 5 min,

4. Resuspend the cells in 5 mL culture medium and plated to 25-cm2 culture flask or dish.

[Passage]

1. Wash the cells twice with PBS and add adequate amount of PBS.

2. Cool the culture for 15-30 min at 4 C.

3. Detach the cells by pipetting in PBS with a glass Pasteur pipette.

4. Collect the cells by centrifugation at 1000 rpm for 5 min.

5. Resuspend the cells in fresh medium and seed onto bacterial grade dishes/flasks.

*MG5 cells stack to the culture substratum very tightly, and this attachment is trypsin-resistant. Even according to the above procedure, only (up to) 70% recovery is expected. In addition, the attachment will become increasingly hard if the cells are cultured for long period in a same dish/flask. We recommend passage interval should not exceed 1 week.

(The cell bank tested the above passage method, but like-macrophages, MG5 adhere very tightly and this passage method did not completely work. The cell bank also tested the pronase digestion, 25 ug/ml pronase in Hanks' BSS for 10 min. and pipetting, but it also appeared not completely to recover the cells.)

[Alternative method for passage.]

Use RepCell (Cell Seed Inc.). Repcell is temperature-responsive cell cutureware, and at 4 C, the cell culture surface lose to support cell attachment. At passage, place the RepCell dishes at 4 C for 30 min to detach the cells.

[Close]

JCRB No.	IFO50520	Cell Name	MG5
Profile	A microglial cell line from p53-deficient mice. Requires conditioned medium of A1 cell line (or of astrocytes) for cultivation of MG5 cells.	Other Name
Animal	mouse	Strain	129/Sv,BDF1,C57BL/6J
Genus	Mus	Species	musclus
Sex		Age	newborn
Identity		Tissue for Primary Cancer	neural
Case history		Metastasis
Tissue Metastasized		Genetics	Established from p53(-/-) homozygote mice (strain; 129/SvJ, BDF1, C57BL/6J).
Life Span	infinite	Crisis PDL
Morphology	microglia-like	Character	microglial cell line from p53-deficient mice
Classify		Established by	Ohsawa, K.
Registered by	Ohsawa,K.	Regulation for Distribution
Comment		Year	1999
Medium	3/7 mixture of DMEM (high glucose) with 10% FBS and A1(IFO 50519)-conditioned medium (CM). The CM is filtered (0.22 um) supernatant of subconfluent A1 culture.	Methods for Passages	Use bacteria-grade dish/flask. For passage, wash the culture twice with PBS and detach cells in PBS by vigorous pipetting with Pasteur pipette.
Cell Number on Passage		Race
CO2 Conc.	5 %	Tissue Sampling	brain
Tissue Type

Detection of virus genome fragment by Real-time PCR
Detected DNA Virus	not tested	Detected RNA Virus	not tested

Link
Cellosaurus(ExPASy) Cellosaurus (ExPASy) is developed by Amos Bairoch of the CALIPHO group at the SIB - Swiss Institute of Bioinformatics as part of the neXtProt project. Cellosaurus is a knowledge database of cell lines with various information summarized.	CVCL_5556

Reference
Pubmed id:11500035	Iba1 is an actin-cross-linking protein in macrophages/microglia. Sasaki Y,Ohsawa K,Kanazawa H,Kohsaka S,Imai Y Biochem Biophys Res Commun. 2001 Aug 17;286(2):292-7
Pubmed id:11406294	Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells. Kawahara K,Gotoh T,Oyadomari S,Kajizono M,Kuniyasu A,Ohsawa K,Imai Y,Kohsaka S,Nakayama H,Mori M Brain Res Mol Brain Res. 2001 Jun 20;90(2):165-73
Pubmed id:10934045	Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia. Ohsawa K,Imai Y,Kanazawa H,Sasaki Y,Kohsaka S J Cell Sci. 2000 Sep;113 ( Pt 17)():3073-84
Pubmed id:9383038	Generation and characterization of a microglial cell line, MG5, derived from a p53-deficient mouse. Ohsawa K,Imai Y,Nakajima K,Kohsaka S Glia. 1997 Nov;21(3):285-98

02232011
02122009
12222008
1159
07172017

Cell No.	IFO50520	Cell Name	MG5
LOT No.	02232011	Lot Specification	distribution
Medium	7:3 mixture of A1-conditioned medium and D.MEM (high glucose) with 10% FBS. [Lot number 02232023 is mislabel. Correct lot number is 02232011.]	Temperature	37 C
Cell Density at Seeding	approx. 1 x 10^5 cells/ml	Methods for Passages	Cells were treated with ice cold PBS (4C for 5 min) and were harvedted by vigoruous pipetting. Split ratio 1:2. Use non-treated (bacterial grade) flasks.
Doubling Time	approx. 82 hrs.	Cell Number in Vial (cells/1ml)	5 x 10^5
Viability at cell freezing (%)	65.4	Antibiotics Used	free
Passage Number	Unknown (15 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	Confirmed as mouse by NP, G6PD, MD.
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information	Lot# is misprinted as 02232023 on vials.

Cell No.	IFO50520	Cell Name
LOT No.	02122009	Lot Specification	distribution
Medium	7:3 mixture of A1-conditioned medium and D.MEM (high glucose) with 10% FBS	Temperature	37 C
Cell Density at Seeding	approx. 1 x 10^4 cells/ml	Methods for Passages	25 ug/ml pronase in Hanks' BSS for 10 min. , and vigorus pipetting.
Doubling Time	approx. 3 days	Cell Number in Vial (cells/1ml)	6.3 x 10^5
Viability at cell freezing (%)	93.7	Antibiotics Used	free
Passage Number	Unknown (6 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	Confirmed as mouse by NP, G6PD, MD.
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	IFO50520	Cell Name
LOT No.	12222008	Lot Specification	distribution
Medium	7:3 mixture of A1-conditioned medium and D.MEM (high glucose) with 10% FBS	Temperature	37 C
Cell Density at Seeding	3 x 10^4 cells/ml	Methods for Passages	25 ug/ml pronase in Hanks' BSS for 10 min. , and vigorus pipetting.
Doubling Time	approx. 3 days	Cell Number in Vial (cells/1ml)	2.5 x 10^5 cells/ml
Viability at cell freezing (%)	88.2	Antibiotics Used	free
Passage Number	Unknown (8 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	Confirmed as mouse by NP, G6PD, MD.
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	IFO50520	Cell Name	MG5
LOT No.	1159	Lot Specification	distribution
Medium	7:3 mixture of A1-conditioned medium and D.MEM (high glucose) with 10% FBS	Temperature	37 C
Cell Density at Seeding	3 x 10^4 cells/ml	Methods for Passages	25 ug/ml pronase in Hanks' BSS for 10 min. , and pipetting.
Doubling Time		Cell Number in Vial (cells/1ml)	8.8 x 10^5
Viability at cell freezing (%)	84	Antibiotics Used	free
Passage Number	Unknown (5 at IFO)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion		Exoteric Gene
Medium for Freezing	10% DMSO, 20% serum (final conc.)-culture medium	CO2 Conc.
Viability immediately after thawing (%)		Additional information

Cell No.	IFO50520	Cell Name	MG5
LOT No.	07172017	Lot Specification	distribution
Medium	7:3 mixture of A1-conditioned medium and D.MEM (high glucose) with 10% FBS.	Temperature	37 C
Cell Density at Seeding	4.3 - 5.9 x 10^5 cells/mL	Methods for Passages	In this lot, RepCell (Temperature-responsive cell cutureware (Cell Seed Inc.)) was used, and the cells were detached by placing the dishes at 4 C freezer for 30 min. Alternative method: Use bacteria-grade dish/flask. For passage, wash the culture twice
Doubling Time	20 - 84 hrs.	Cell Number in Vial (cells/1ml)	2.0 x 10^6
Viability at cell freezing (%)	87.7	Antibiotics Used	free
Passage Number	Unknown (6 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - DMEM (high glucose)	CO2 Conc.	5%
Viability immediately after thawing (%)	87.7	Additional information

JCRB Cell Bank

IFO50520 MG5

Cell information

LOT Information