Olfactory bulb sheath-forming cells
Olfactory bulb sheath-forming cells
This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)
Operation method
Program 23.20 Olfactory bulb sheath-forming cells
Materials and Instruments
L-polylysine type I collagenase HBSS DMEM FBS DMEM BS monoclonal antibody secondary antibody Sprague-Dawley rat Move Preparation of isolated OEC suspensions For more product details, please visit Aladdin Scientific website.
Leibowitz L-15 Culture Solution 70% Ethanol
Culture flasks Petri dishes Coverslips Petri dishes 15 ml conical polypropylene tubes with lids No. 7 curved and straight forceps Round-bottomed polypropylene tubes with lids Curved dissecting scissors Scalpels Plates and needles for excision Fluorescence-activated cell sorter
1. Rats are killed by decapitation (Sprague-Dawley rats: 6-8 d postnatal.). Although OEC can be isolated from rats of any age, more OEC is obtained from neonatal rats than from older rats). Cells were taken from 15-20 rats and sorted by FACS (Fluorescence Activated Cell Sorter (FACS; B-D Biosciences)) for 2-3 h. Enough OEC were sorted to cover 10-15 coverslips with 10,000 cells per coverslip.
Culture flasks, dishes, and coverslips: pre-coated with 13.3 mg/ml L-polylysine
(a) Incubate for 1~24 h. The cells were incubated with 13.3 mg/ml L-polylysine;
(b) Wash with D-PBSA;
(c) Air dry before use.
2. Position the dorsal part of the head on the excision plate and spray the head with 70 % ethanol.
3. soak all instruments in 70 % ethanol before use and shake the instruments to dry them.
4. The skin is removed with sharp curved scissors and a circular incision is made to remove the skull cap, revealing the brain and the two olfactory bulbs at the tip of the nose. 
5. The olfactory bulbs were gently removed from the brain with curved forceps and placed in a Petri dish with a drop of L-15 (containing gentamicin).
6. Cut the olfactory bulbs into small pieces with a sterile surgical blade.
7. Place the olfactory bulb pieces into a vial containing 1 ml of Collagenase Reservoir Solution and 1 ml of L-15.
8. Incubate the olfactory bulb pieces at 37°C for 30-45 min.
9. Centrifuge the suspension (1000 r/min for 5 min) and aspirate the supernatant.
10. Suspend the olfactory bulb tissue with 1 ml of Ca2+ and Mg2+ free HBSS.
11. Glial cells are easily damaged. Therefore, the olfactory bulb tissue must be gently dispersed to form a single-cell suspension, taking care not to create air bubbles. Disperse the olfactory bulb tissue through a 19 G hypodermic needle and then through a 23 G hypodermic needle.
12. Add 5 ml of Ca2+ and Mg2+ free HBSS and centrifuge the suspension (1000 r/min, 10 min ). Then, the supernatant was aspirated and the cells were suspended in DMEM-FBS (DMEM (GIBCO) with 5 % FBS) at a concentration of 5 × 106 cells/ml.
Sorting OFC by FACS
In order to remove as many O4+ oligodendrocytes as possible from the sorted cells, it is preferable to use secondary antibodies against galactosylceramide (anti-mouse IgM-fluorescein and anti-mouse IgG3-phycoerythrin (Southern Biotechnology Associates or Cambridge Biosciences)) (anti-GalC ) [ Ranscht et al. 1982; Barnett et al. 1993 ] label cells to discriminate between oligodendrocytes and OEC. in this regard, two-color sorting can be performed, and unwanted cells labeled with both antibodies can be ignored. Sort with FACSVamage for 1 h .
13. After dispersing the tissue, 5×106 cells are placed in a 15 ml centrifuge tube, and the cells are suspended in a mixture of 500 μl of O4 and ami-GalC antibody hybridoma supernatant and incubated for 30-45 min at 4°C. The total number of cells will be more than 5×106, and the number of cells will be more than 5×106. The total number of cells will be more than 5×106, so enough centrifuge tubes should be prepared. If the hybridoma supernatant does not contain a high concentration of antibody, increasing the cell number will result in a significantly lower percentage of O4+ cells. A similar analysis is used for anti-GalC hybridoma supernatants. Therefore, titration of the antibody by FACS analysis is often required prior to use.
14. Cells are washed twice with DMEM-FBS and then suspended in 500 μl of DMEM-FCS containing goat anti-mouse IgM-fluorescein (1:100) and goat anti-mouse IgG3-phycoerythrin (1:100) and incubated at 4°C for 30 min.
15. Cells were washed twice by centrifugation with 15 ml DMEM-FBS. The cells were then incubated with ice-cold DMEMFBS (DMEM (GIBCO ), supplemented with the following (Bottenstein and Sato, 1979): 25 mmol/L (4.5 g/L) glucose, 25 μg/ml gentamicin (Invitrogen), 0.0286% (V/V) BSA pathocyte ( MP Biomedicals), 2 mmol/L glutamine, 10 μg/ml bovine membrane insulin, 100 μg/ml human transferrin, 0.2 μmol/L progesterone, 0.10 μmol/L L-putrescine, 0.45 μmol/L L-thyroxine, 0. 224 μmol/L selenium, and 0.49 μmol/L 3,3,5-L-triiodo thyrotropine. (All reagents except gentamicin and BSA pathocyte were from Sigma.) Cells in the sorting tubes (2085, BD Biosciences) were suspended at a density of 2 × 106 cells/ml. the sorting tubes were placed on ice. FACS images of antibody-labeled cells were stable for at least 3-4 h when cells were placed on ice.
16. This procedure describes how the FACStar Cell Sorter sorts cells. However, any type of cell sorter should produce similar results. Align the laser with the 488 nm excitation light. Fluorescence compensation must be set to remove excess fluorescence from each fluorescence source. Prior to sorting cells labeled with the secondary antibody alone, control cells should be analyzed to facilitate interpretation of non-specific labeling and removal of non-specific fluorescence by reducing the gain of the photomultiplier tube.
17. set the cell sorting window near O4+GalC-. the average percentage of O4+ olfactory bulb cells should be 10% ± 1% .
18. Sort O4+GalC- cells into a round-bottomed polystyrene container containing 4 ml of DMEM-FBS.
19. After sorting, transfer the sorted cells into 15 ml centrifuge tubes. Wash the sorting tube with culture solution, add the wash solution to the centrifuge tube, and then add DMEM-FBS. centrifuge (100 g, 15 min).
20. Aspirate the supernatant and suspend the cells with DMEM/BS. Desirable OEC viability and growth were obtained when diluted with DMEM/BS:ACM (1:1) [ Barnett et al., 1993].ACM was collected from purified cortical astrocyte monolayers after 48 h of incubation with DMEM/BS [ Noble and Murrey, 1984].