hi all , whom have been on the same view as my questions ,
1. could you help to suggest for me some methods to improve the proliferation of keratinocytes (HaCaT cells). my problem now is that the differentiation seems to be predominant meanwhile the amount of new cells keratinocytes not as many as my expectation
2. in accordance with cells number , is there any kit or agent which can enhance the growth rate of keratinocytes (HaCaT cells), a little faster . currently , my cells need at least 4 days .
many thanks for your time and concern ,
Hello, An
You look a t this article:
Article Growth and Differentiation of HaCaT Keratinocytes
2.1 Cell Culture Components 1. 3.0 mM calcium chloride (100 stock): Weigh 333 mg of calcium chloride (Sigma-Aldrich; 99.99 % anhydrous powder) and mix into 1 l of cell culture-grade water. Stir until dissolved, and then filter sterilize directly into a sterile 1,000 ml bottle using the Stericap™ Plus Universal Bottle-Top Filter Device (Millipore; 0.22 μm pore). Store at 4 C. 2. 280 mM calcium chloride (100 stock): Weigh 31.07 g of calcium chloride (Sigma-Aldrich; 99.99 % anhydrous powder) and mix into 1 l of cell culture-grade water. Stir until dissolved, and then filter sterilize directly into a sterile 1,000 ml bottle using the Stericap™ Plus Universal Bottle-Top Filter Device (Millipore; 0.22 μm pore). Store at 4 C. 3. Low-calcium fetal bovine serum (FBS): To remove endogenous calcium in the FBS (BenchMark™ Fetal Bovine Serum; Gemini Bio Products) it must be treated with Chelex 100 resin (BioRad; catalog number 142-2832). Distribute 50 ml aliquots of FBS into sterile, 50 ml conical tubes, and add 0.38 g of Chelex to each tube. Incubate tubes for 1 h at 4 C on a tube rotator and then use immediately for the low-calcium and high-calcium media (see Note 1). 4. Low-calcium (0.03 mM) growth medium: To 870 ml of DMEM add 20 ml of 200 mM L-glutamine, 10 ml of 3.0 mM calcium chloride solution, and 100 ml of the lowcalcium FBS. Swirl briefly to mix components, and then filter directly into a sterile 1,000 ml bottle using the Stericap™ Plus Universal Bottle-Top Filter Device (Millipore; 0.22 μm pore). This removes the Chelex resin and ensures sterility of the combined solution. Store at 4 C. 5. High-calcium (2.8 mM) growth medium: To 780 ml of DMEM add 20 ml of 200 mM L-glutamine, 10 ml of 280 mM calcium chloride solution, and 100 ml of the lowcalcium FBS. Swirl briefly to mix components, and then filter directly into a sterile 1,000 ml bottle using the Stericap™ Plus Universal Bottle-Top Filter Device (Millipore; 0.22 μm pore). This removes the Chelex resin and ensures sterility of the combined solution. Store at 4 C. 6. 0.25 % Trypsin, 1 g/l EDTA (Hyclone). Van G. Wilson 7. Phosphate-Buffered Saline (PBS), pH 7.4 (Life Technologies). 8. DMEM (High Glucose, no Glutamine, no Calcium; Life Technologies). 9. 200 mM L-glutamine (Atlanta Biologicals).
Or this article:
Article Proteomic characterization of HaCaT keratinocytes provides n...
Cell culture and treatments
HaCaT cells were seeded in T75 tissue culture flasks (Corning, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), Glutamax™ Supplement, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Media components were purchased from Gibco, USA. Cells were maintained at 37 °C and in a humidified 5% CO2 atmosphere. The medium was changed every other day.
Hi An Nguyễn Hoàng
In addition to Alexandr Chernov brilliant response, remember to maintain your cell lines between 40-80% as a rough guide - any less and your cells will be in lag phase and not proliferate optimally while any more and your cells will suffer a lack of resources and contact inhibition.
dear Dr Alexandr Chernov , I have read carefully all of references you have mentioned. probably I will apply these components for some experiments to find the best combination for my cell stock . actually , I have already checked all of the conditions for cells storage also and maintain procedure is perfect , CO2 always 5%. Again thank you for your recommendations , a lot of references but it is better to read some of suggestions. sincerely, wish you health and joyfulness
dear Dr Jack Hopkins , yes absolutely I agree with your suggestion . thank you for your emphasized view as my observation. It is intensively concerned with the confluency I have cultured. and it seems like cells locate into cluster and grow well in cluster together. so amazing , as if contact inhibition or activation as you have mentioned. it is my thankfulness , sincerely , wish you health and joyfulness
thank you so much for all of your suggestion , the cell line culture becomes quite good. thank you again , most sincerely,
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