Method for separating and culturing hippocampal nerve cells and special culture solution thereof
A technology for hippocampal neurons and cell suspensions, applied in the field of hippocampal neuron cell separation and primary culture methods and reagents, can solve the problems of easy to increase bacterial contamination, lack of suitable, difficult to obtain, etc., to improve in vitro survival rate and digestion efficiency Improve and increase the effect of physiological breathing
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Embodiment 1
[0041] Bifidobacterium longum (Bifidobacterium longum) LJM002 of the present invention has been stored in the General Microbiology Center of China Microbiological Culture Collection Management Committee on May 25, 2011, which is referred to as CGMCC (Address: No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing) No. 3 Institute of Microbiology, Chinese Academy of Sciences, zip code 100101) is preserved, the classification is named Bifidobacterium (Bifidobacterium), and the preservation number is CGMCC No.4900.
[0042] The Bifidobacterium longum LJM002 of the present invention was isolated from the feces of a healthy young man in Zhejiang Province.
[0043] Bifidobacterium longum LJM002 bacterial strain of the present invention has following microbiological characteristics:
[0044] (1) Colony morphology: the colony of Bifidobacterium longum LJM002 strain on the plate is off-white or milky white, opaque, shiny, smooth, raised, soft in texture, with neat edges, and 1-...
Embodiment 2
[0051] Reagent purchase:
[0052] DMEM / F12 nerve basal medium, Neurobasal medium and B27 were purchased from Gibco; polylysine, fetal bovine serum (FBS), trehalose and L-glutamine were purchased from Sigma, USA; penicillin and streptomycin were mixed Solution (double antibody) was purchased from HyClone Company in the United States.
[0053] Reagent configuration:
[0054] (1) The D-Hank's solution is obtained through the following steps: NaCl 18.0g, KC 10.4g, NaCl 2 HPO 4 12H 2 O0.12g, KH 2 PO 4 0.06g, NaHCO 3 0.35g; Dissolve each component in approximately 500mL triple-distilled water and mix well, add triple-distilled water to make up to 1000mL, adjust the pH value to 7.2-7.4, subpackage, autoclave, subpackage, and store at 4°C for later use.
[0055] (2) The rinse liquid is obtained through the following steps: dissolve 2g trehalose, 3g glucose and 10mL double antibody in 100mL D-Hank's solution, mix well, add D-Hank's solution to 1000mL, 0.4 Dissolve in hydrogen u...
Embodiment 3
[0066] The method for separating and primary culture of SD rat hippocampal neurons comprises the following steps:
[0067] Use the reagents prepared in Example 1 and Example 2 and configure the reagents.
[0068](1) Rinsing: Take the hippocampus tissue of the isolated mammal, put it in the rinsing solution in an ice bath, remove red blood cells, capsules and connective tissue, and rinse it 2 to 5 times with the rinsing solution;
[0069] (2) Digestion: cut the hippocampal tissue rinsed in step 1 into a diameter of 1mm 3 For small pieces, use 5 times the tissue volume of digestion solution at 37°C for 5-10 minutes to organize into a porridge-like shape, stop digestion with cell seeding solution, and gently pipette until the tissue piece is 10 times to disperse the cells.
[0070] (3) Preparation of cell suspension: Collect the initial cell suspension after digestion in step 2, filter through a 200-mesh cell sieve, centrifuge at 800-1000 rpm at 4°C for 5-10 minutes, discard the...
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