Cell Use Instruction - HaCaT Cell Line

Product Info

Catalog YC-A018

Cell line HaCaT

Morphology Epithelial-like, adherent Passage ratio 1：3~1：5

90%DMEM+10%FBS
Culture method
Ubigene didn't use P/S. But client could use P/S after cells grow in good
condition after thawing.

Cryopreservation 50%DMEM+40%FBS+10%DMSO
solution

Special Note

STR Authentication

STR Info (Sample Cell) STR Info (Cell bank)

Loci Sample Cell Line: HaCaT Cell Line: HACAT

Allele1 Allele2 Allele3 Allele1 Allele2 Allele3

D5S818 12 12 12 12

D13S317 10 12 10 12

D7S820 9 11 9 11

D16S539 9 12 9 12

VWA 16 17 16 17

TH01 9.3 9.3 9.3 9.3

AMEL X X X X

TPOX 11 12 11 12

CSF1PO 9 11 9 11
* STR authentication data of this cell line matches with that of cell lines sourced from ATCC, DSMZ, JCRB, and
RIKEN databases.
Conclusion: The STR identification of this cell is correct.

Cultured cell morphology

Cell Reception

Cryopreserved cells:

In the case of cryopreserved cells transported with dry ice, upon received, immediately transfer to liquid

nitrogen for storage or store briefly at -80°C freezer, or proceed directly to cell thawing. Upon cell

thawing, please count the cell number and cell viability and take some photos of the cells under

different magnification (e.g. at 100x and 40x) as the records.

Notice: Upon received, please ensure to takes photos of the package, including dry ice and the tubes,

and contact us within 24 hrs if any abnormalities such as dry ice has ran out, the cap of the cryovial is

dislodged, broken and the cell is contaminated.

Cell Thawing

1) Preparation: warm up the complete culture medium in 37°C water bath for 30 mins. Transfer the

cryopreserved vial from liquid nitrogen to - 80°C freezer, and leave for several minutes to volatilize
 residual liquid nitrogen;

2) Inside the ultra-clean bench, pipet 6-7 mL of complete medium into a 15 mL centrifuge tube;

3) Take out the cryopreserved vial from - 80℃ freezer and leave in dry ice temporarily, shake slightly

before thawing to remove residual dry ice and liquid nitrogen. Then hold the cap with forceps,

quickly thaw cells in a 37°C water bath by gently swirling the vial (Note: keep the cap out of the

water). In about 1 minute, it would completely thaw;

4) Inside the ultra-clean bench, sterilize the outer surface of the vial by wiping with an alcohol cotton

pellet and leave it to dry. Transfer the thawed cells to the prepared centrifuge tube (step 2) by

pipette, close the lid, and centrifuge at 1100 rpm for 4 mins at room temp to collect the cells;

5) Inside the ultra-clean bench, carefully remove and discard the supernatant. Resuspend cell pellet

with 1mL of fresh complete medium and then transfer to a T25 flask (or 6 cm culture dish)

containing 4 mL of complete medium, label the flask with cell name, date and passage no., incubate

the flask in a 37℃, 5%CO2 incubator.

Note: Please do not thaw the cells directly to a T75 flask or 10 cm culture dish.

Cell Passaging

1) As long as the cells are 80%-90% confluent, it is ready to passage. Inside the ultra-clean bench,

remove and discard the medium from the flask and briefly rinse the cell 1-2 times with 1×PBS (2-3

mL for T25 flask, 4-5 mL for T75) to remove residual medium and serum;

2) Add the corresponding volume of trypsin solution (see below table 1 for details) and allow trypsin

completely cover the cells, place the flask into the incubator and incubate for 1-2 mins (If cells are
 hard to digest, allow appropriate extension of incubation), until the majority of the cells become

round and non-adherent as observed under the microscope, a large number of cells detached from

each side when gently shaking and tapping the flask, terminate trypsin digestion immediately;

3) Add complete medium to stop digestion, the volume is 2 times of trypsin. Then gently pipet the cells

several times to allow all cells to be completely detached from the flask;

4) Transfer the cell suspension with a 10 mL pipette into a 50 mL centrifuge tube, rinse the residual

cells from the flask using appropriate volume of PBS , then collect and put them together to the

centrifuge tube;

5) Centrifuge at 1100 rpm for 4 mins at room temp. After centrifugation, remove and discard the

supernatant and resuspend the cells with 2 mL of complete medium;

6) Cells need to be passaged at appropriate passage ratio, 1:3 for the first passage, increasing the

passaging ratio if the cells are grown to confluence within two days, or decreasing the passaging

ratio if the cells are not grown to confluence in 3-4 days.

Table 1. Volume of Trypsin solution added to different size of culture plates/flasks

Size of culture plates/flasks Trypsin Volume added

6-well plate 0.5 mL

T25 1 mL

T75 2-3 mL

T175 3-4 mL

Cell cryopreservation

1) Same as procedures of cell passaging, inside the ultra-clean bench, digest the cells to a single-cell
 suspension, and terminate digestion by adding complete medium. All liquid is transferred to a 50

mL centrifuge tube;

2) Mix well by pipetting and take 20 μL for cell counting;

3) Centrifuge at 1100 rpm for 4 mins at room temp. After centrifugation, remove and discard the

supernatant, and resuspend the cells with 1-2 mL of 4℃ pre-cooled cryopreservation medium (use

the one you usually use in lab, or any commercial cryopreservation solutions are fine), then add

cryopreservation medium to adjust to the required density (1x106-1x107cells/mL);

4) Aliquot the cell suspension to cryovials as 1 mL/tube, close the lid tightly, and the cryovials should

be labeled with the cell name, source, cell passage number, and date of cryopreservation in

advance;

5) Place the cryovials in 4°C pre-cooled Freezing Container, then put the container in -80℃ freezers

within 15 mins after cell cryopreservation;

6) Stay overnight, transfer the cryovials to liquid nitrogen for long-term storage.