Subculture of mesenchymal stem cells
Reagents and materials:
1. Complete medium: α-MEM: α-low-limit basal medium containing glutamine, no nucleotide or deoxynucleotide; addition: 20% additional L-glutamine 2mmol/L, hybridized by FBS Tumor purification, non-heat inactivation, penicillin 100 U/ml, streptomycin 100 μg/ml. Filtration sterilization. Store at 4 ° C for no more than 2 weeks;
2. PBSA;
3. Trypsin / EDTA;
4. Polypropylene centrifuge tube, 15ml and 50ml;
5. Plastic tissue culture dish, diameter 15cm;
6. Plastic micropipette tip, 10-1000μl;
7. 0.85% trypan blue salt solution;
8. Micropipettes;
9. Improved Neubauer blood cell counter
experimental method:
1. Treat small, adherent, spindle-shaped fibroblast-like cells in cultured MSCs cells at 60% confluence. If the monolayer cells are sparse, continue to rinse and change the medium;
2. Digest the monolayer cells as follows;
(1) After washing the monolayer cells with 20 ml of pre-warmed PBSA, add 5 ml trypsin / EDTA;
(2) The culture dish was placed at 37 ° C for 2 min, and the monolayer cells were observed under a 10× magnification field of view. The adherent cells should be detached from the plastic bottle;
(3) Place the culture dish at 37 ° C for 2 min and observe again. Repeat observation until 90% of the MSCs have been detached from the culture flask;
(4) Add 5 ml of CCM, transfer 10 ml of the suspension to a 15 ml centrifuge tube, and centrifuge at 500 g for 10 min;
(5) After centrifugation, discard the supernatant and resuspend the pellet with 1-2 ml of pre-warmed PBSA. If necessary, mix the multi-cell suspension with only one cell count;
3. 10 μl of the cell suspension was added to 10 μl of trypan blue and counted using a hemocytometer. A suitable cell suspension concentration is (2-5) x 105 cells/ml, and the survival rate needs to be higher than 80%. Early culture suspensions for passage after trypsin digestion may also be assayed for colony forming units (CFU), cryopreserved or differentiated;
4. Inoculate cells at a density of 50-100 cells/cm into the culture dish, maintaining a low density to ensure rapid self-renewal and pluripotency. Resuspend MSCs with preheated CMM at a density of 7 x 103 (final concentration 50 cells/cm2) to 1 x 104 (final concentration 100 cells/cm2);
5. Prepare an appropriate number of 15cm culture dishes and add 25ml of preheated CMM;
6. Inoculate 1 ml of the cell suspension obtained in steps 2 and 3. Slide the plate back and forth (do not circle) to evenly distribute the cells. The culture dish is placed in an incubator and labeled as a generation of cells;
7. After 2-3 days of culture, observe and evaluate the morphology. MSCs should be small, spindle-shaped, frequently-refractive doublets. This is a well-cultured MSCs;
8. Aspirate the medium from the Petri dish, rinse with 20 ml of pre-warmed PBSA, and add 25 ml of pre-warmed fresh CMM;
9. The number of dishes required for each passage depends on the number of cells that can be inoculated. The above volume in the protocol is suitable for MSCs to inoculate a single 15 cm dish. If necessary, expand to inoculate multiple culture dishes according to this example;
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