Here, our "bullet-proof" isolation protocol, for detailed information on chemicals etc. click here!

Add 20 ml of Tris/HCl (50 mM, pH 9,5) to 2 Petri-dishes each (subtraction, Æ 150 mm, bacterial culture quality, Falcon), shake until surface is covered by liquid, add 60 µl affinity-purified goat anti-rabbit IgG to each dish, incubate overnight at 4°C or 2 h at room temperature

Add 10 ml of Tris/HCl (50 mM pH 9,5) to 1 Petri-dish (selection, Æ 100 mm, bacterial culture quality, Falcon), shake until surface is covered by liquid, add 30 µl affinity-purified goat anti-mouse IgM to the dish, incubate overnight at 4°C or 2 h at room temperature

After incubation, wash all plates 3 times with PBS, add 15 ml 0.2% BSA in D-PBS (Dulbecco’s PBS containing 1g/l Glucose, 36 mg/l Sodium pyruvate, Gibco) to the subtraction plates and 5 ml to the selection plate

Add 5 ml T11D7 hybridoma culture supernatant (containing approx. 20-50 µg/ml anti-Thy1 IgM) to the selection plate, incubate at room temperature for at least 2 hrs.

Dissect retinae of 5-10 rats (postnatal day 5-7) and collect them in D-PBS, clean by removing adhering tissue and lens material

Incubate 165 U Papain in 5 ml D-PBS for 5 min at 37°C for dissolution, sterile filter, add 1000 U DNase (endconc. 33 U/ml Papain, 200 U/ml DNase)

Transfer retinae into Papain solution, incubate at 37 °C for 30 min, invert tube carefully (!) after 10 and 20 min

Remove supernatant from papain-treated retinae and add carefully (!) 4 ml of 0.15% ovomucoid solution, wait until retinae settle and remove supernatant

Add 80 µl anti-macrophage serum and 2000 U DNase to 6 ml 0.15% ovomucoid solution

Add 1.5 ml of this solution to retinae

Pipet retinae 5-10 times up and down with blue tip (1ml) cut at 8 mm from top. Pipetting velocity approx. 1-2s per up- or down-stroke

Let cells settle, transfer supernatant containing dissociated cells to separate tube

Pipet retinae 5-10 times with tip cut at 5 mm from tip, let cells settle etc.

Repeat trituration with intact blue tip, until tissue is completely triturated

Wait 5 min to allow for antibody binding

Centrifuge cell suspension for 13 min at 129 g, discard supernatant and resuspend in 6 ml 1% ovomucoid solution

Centrifuge cell suspension for 13 min at 129 g, discard supernatant and resuspend in 10 ml 0.02% BSA in D-PBS

Wet nylon mesh (sterilized by UV-light) with 0.02% BSA in D-PBS, place it as a funnel on top of a 50 ml tube and filter cell suspension

Fill cell suspension to 15 ml with 0.02% BSA in D-PBS

Remove BSA solution from first subtraction plate (Æ 150 mm), pour cell suspension on plate and incubate at room temperature for 36 min, shake gently every 12 min

Remove BSA solution from second subtraction plate (Æ 150 mm), shake first panning plate vigorously to resuspend unbound cells, pour cell suspension onto second plate, carefully transfer any residual liquid from 1st plate and incubate at room temperature for 33 min, shake gently every 11 min

Wash selection plate 4 times with D-PBS, shake second subtraction plate vigorously to resuspend unbound cells, filter cell suspension through wetted Nitex mesh (as above), pour filtered cell suspension on selection plate and incubate at room temperature for 45-60 min, shake gently every 15 min

Mark place of pouring to minimize shearing

Wash selection plate with D-PBS up to 10x by gently swirling, dump D-PBS directly, but carefully (!) at marked place

Confirm absence of non-adhering cells under microscope, if necessary wash more often

Wash selection plate with EBSS (Earl's balanced salt solution w/o Ca and Mg, pre-equilibrated in incubator at 5% CO₂; Attention: EBSS is carbon buffered!)

Add 4 ml pre-equilibrated EBSS and 200 µl trypsin solution (2.5%, Sigma) to plate and incubate at 37° at 5% CO₂ for 10 min

Add 4 ml FCS (30% in D-PBS) to plate, pipette cell suspension up and down on the inclined plate to remove cells, especially around the rim and transfer cell suspension to Falcon tube. Be careful not to impose too much mechanical stress on cells!

Repeat previous step, until all cells are removed (confirm under microscope)

Invert tube several times, take sample of 50 - 100 µl for cell counting in hemocytometer and centrifuge tube for 13 min at 129 g

Resuspend cell pellet in medium at 1000 cells / µl

Plate cells at appropriate density (e.g. ca. 80000 cells / 35 mmm dish) in dishes previously coated with 5 µg/ml PDL (MW 30-70000; in H₂O for 1 h at room temperature, washed 3x with H₂O and air-dried)

Culture in chemically defined medium

Feed cells every 2-3 days by placing half of the medium with fresh medium NB⁺_conc containing all supplements at double concentration

Viability reaches stable level after 5 days (about 30-50%)