Keloid cell line subculture protocol

A. Equipment:
1. 37 o C incubator with humidified atmosphere of 5% CO 2 (Panasonic; Serial no
15070066);
2. Laminar flow hood BSC-4 (Taiwan brand);
3. Hemocytometer (Neubabuer);
4. Centrifuge (Kubota 2100; Serial no B427-337);
5. Water bath (Cherng Huei Instrument);
6. Pipette P1000, P200 and/or P100, P20 and/or P10 (NICHIRYO, Thermo
Scientific);
7. Liquid Nitrogen Tank (International Cryogenics, Inc; Serial no IC
35RX);
8. Microscope (Olympus CXX31; Serial no IX2-SLP);
9. Alcohol lamp (the inside is filled with industrial alcohol, at least 90% EtOH,
transparent or pink-colored);

B. Materials needed:
1. Cell culture treated flasks, plates or dishes;
2. Disposable, sterile 15-mL tubes, 50-mL tubes;
3. Disposable, sterile tips;
4. Disposable, sterile 1.5 ml microcentrifuge tubes;
5. Kimwipes Delicate Desk Wipers (Kimberly-Clark™ Professional
KC34120)

C. Reagent:
1. Balanced salt solution Dulbecco’s phosphate-buffered saline (DPBS)
(Gibco; Dulbecco’s phosphate-buffered saline; Cat: 14190205);
2. Dissociation reagent Trypsin EDTA (Gibco; 0.5% Trypsin-EDTA (10X);
Cat: 15400-054);
3. Complete growth medium Dulbecco’s Modified Eagle Medium
containing high glucose (DMEM); (Corning; Dulbecco’s Modified Eagle
Medium; Cat 00621008);
4. Fetal bovine serum (FBS) (Gibco; Fetal bovine serum; Cat: 10437-077);
 5. Penicillin – Streptomycin (100X) (Caisson; Cat: PSLC0140): contains
10,000 U/ml of penicillin and 10,000 µg/ml streptomycin in a 0.9% saline
sterile-filtered solution.
6. 75% ethanol;

D. Solution preparation
1. Dissociation reagent 0.05% Trypsin EDTA, pre-placed at room
temperature;
2. Complete growth medium: 10% FBS-DMEM-high glucose-1%PS
medium
40ml DMEM + FBS (4.5ml) + PS (0.4 ml)
45ml DMEM + FBS (5ml) + PS (0.5 ml)
3. 75% ethanol (Ethanol 99% diluted to 75% with ddH 2 O);

E. Cell subculture procedure:

Notes: All solutions and equipment that come in contact with the cells must be
sterile. Always use proper sterile technique and work in a laminar flow hood.
The volume of medium and growth area in different dish size will be showed in
the table A below;
Step 1: View cultures using an inverted microscope to assess the degree of
confluency and confirm the absence of bacterial and fungal contaminants; The
cell was confluent around 90%, it is supposed to culture today (2021/08/09)
 Step 2: Turn on UV light for 30 minutes in laminar flow.
 Step 3: After 30mins, turn off UV light of laminar flow; warm up medium for
5mins and place medium ready in the hood.
 Step 4: Sterilize the cap of medium tube using alcohol lamp.
 Step 5: Take out the cells from incubator and place into laminar hood; then
remove and discard the spent cell culture medium from the culture vessel;
 Step 6: Pipette trypsin-EDTA onto the washed cells using 1 ml per T25 flask,
tap on side or well bottom, then return to the incubator and leave for 3 minutes
(no more than 5 mins);
For further trypsin-EDTA volume needed for different sized wells, we can
follow the table A below;
 Step 7: During the time waiting for trypsinization, taking new 6cm/10cm dish
add 5ml medium into the incubator;
 Step 8: After 3 minutes, examine the cells using an inverted microscope to
ensure that all the cells are detached and floating. The side of the flasks may be
gently tapped to release any remaining attached cells.
 Step 9: Add 3 ml DMEM (10% FBS) to neutralize the trypsin-EDTA activity;
 Step 10: Extract 10µl to calculate cell concentration; Add the rest to 15ml
centrifuge tube at 1200rpm for 5 minutes;
 Step 11: During the time waiting for centrifugation, counting cell number;
 Step 12: After 5-minute centrifugation, remove supernatant and leave pellet;
 Step 13: Add 1ml medium back to dissolve,
 Step 14: Calculate the cells by adding the cell solution to the 10cm dish that
have prepared in step 7.

Table A: The volume of medium and growth area and trypsin-EDTA volume
needed for different wells

Culture Growth Volume of Seeding Cells at confluent Volume of

vessel area (cm 2) medium density trypsin-
(ml) EDTA(ml)
Flask
T25 25 15 2.5 x 10 5 8.6 x 10 5 0.6 – 1.2
Dish
6 cm 21.5 8-10 1.7-2.5 x 10 5 8.6 x 10 5 0.6 – 1.2
10cm 56.7 10-15 4.5-6.8 x 10 5 2.2 x 10 6 1–2
Cluster
plates
6-well 9.6 4 7.6-11.5 x 3.8 x 10 5 0.3 – 0.5
10 4
12-well 3.5 2 2.8-4.2 x 10 4 1.4 x 10 5 0.2 – 0.4
24-well 1.9 1 1.5-2.2 x 10 4 7.6 x 10 4 0.1 – 0.3
48 -well 1.1 0.5 8.8 x 10 3 – 4.4 x 10 4 0.1 – 0.2
13.2 x 10 4
96-well 0.32 0.1 2.5-3.8 x 10 3 1.2 x 10 4 0.05 – 0.1
How to count the cells
Step 1: Clean the hemocytometer and place the coverslide on top;
 Step 2: Add viability dyes to the sample if desired (1:1 dilution);
 Step 3: Pipette 10 µl of sample and inject into a chamber;
To avoid incorrectly counting, make sure not to form air bubbles.
 Step 4: Start to count the cells:
Count the number of cells present on each of the partitions in the four corners of
the counting chamber (1 mm 2 partitions) with a microscope set at 100x
magnification. For cells located on lines, determine which sides are to be
counted in advance. For example, you may count cells present on the upper and
right side lines, but not the bottom and left sides.

 Step 5: Calculate number of cell as follows.

Total number of cells∈4 squares
Number of cells (cells/ml) = x 104 x dilution ratio
4
Total number of cells = Number of cells x volume of cell suspension