Hybridoma cells clones

General execution:

1. ELISA of the supernatants
2. Antibody staining on cryostat section (7 dpa tail sections) of the positive clones from the ELISA
3. Cloning of a possible candidate
4. ELISA of clone which are grown from a single cell
5. Antibody staining on cryostat section (7 dpa tail sections) of the positive clones from the ELISA
6. Expand one of the clones which was positive on the last ELISA and the Antibody staining
7. Freeze enough cells and supernatant
8. Purify Antibodies

ELISA (step 1 & 4)

Buffer & Material

• Using costar 3369eia/ria plate
• Coating buffer: 0.1M NaHCO3, ph 9.3 (adjusted with 1M NaOH)
• Wash buffer: 0.05% Tween20 in 1x PBS
• 10x blocking buffer 0.5% Tween20, 2% casein, 10x PBS

Procedure

1. Coating the plate a. Antigen at 5 µg/ ml in coating buffer (GST – protein or capture AB: donkey anti mouse IgG Jackson 715-005-150)

b. Add 50 µl/well and incubate overnight at 4 degree

1. Wash plate 3 times over 10 min with wash buffer
2. Block for 1 hour @RT with blocking buffer
3. Wash the plate 3 times over 10 min with wash buffer
4. Add samples in 50µl and incubate for 1 hour @ RT
5. Wash plate 5 times over 10 min with wash buffer
6. Add detection antibody at 1: 10.000 and incubate for 1 hr @ RT (HRP-goat anti- mouse Jackson 115-035-062)
7. Wash 5 times over 10 min with wash buffer
8. Add 100 µl of substrate (ABST (NH4)2 Sigma A3217 (100 ml liquid); A1888 powder)
9. Measure absorbance at 405 nm (or 620 nm) after colour develops

Antibody Staining (step 2 & 5)

a) Preparation of animal tissue

a.1) Gelatin embedding

Material

- 0.03% Benzocain for 1l: - 50 mls 400% Holtfredter’s - 50 mls 10x TBS pH * - 897 mls di H2O - 3 mls 10% benzocaine (is usually in Ethanol dissolved)

- 0.1M sodium phosphate buffer - 2% PFA (in 0.1% sodium phosphate buffer) - 10% sucrose (in 0.1M sodium phosphate buffer) - 20% sucrose (in 0.1% sodium phosphate buffer) - 3,75% gelatine (Merck Bloom 80-120)in 20% sucrose (in 0.1% sodium

 phosphate buffer)

- 7,5% gelatine in 20% sucrose (in 0.1% sodium phosphate buffer) - Plastic mould - Dry ice

Procedure

- Anesthetise the animals (small axolotl with 0.01% benzocain) - Cut the tail in 2% PFA for few hours @RT or o/n at 4 degree - Wash 3 times for 10 min with 0.1 M sodium phosphate buffer to remove

 the PFA

- Infiltrate the tissue with 10% sucrose (in sodium phosphate buffer) @ 4

 degree on a rocker until the tissue has sunk down (a proximally over the 
 day)

- Infiltrate the tissue with 20% sucrose (in sodium phosphate buffer) @ 4

 degree on a shaker o/n until the tissue sank down

- Incubate the tissue in 3.75% gelatine (Merck (bloom) 80 – 120!) in 20%

 sucrose (in 20% sucrose in sodium phosphate buffer) o/n @ 37 degree

- Incubate the tissue in 7.5% gelatine (in 20% sucrose in sodium

 phosphate buffer) for at least 30’ @ 37 degree

- Embed the tissue in 7.5% gelatine (in 20% sucrose in sodium phosphate

 buffer) in plastic mould in the desired orientation (store @ -80 degree)

- Sections: -30 degree Object, -25 degree knife

Or

a.2) Tissue – Tek O.C.T compound

- (Infiltrate the tissue with 10% sucrose (in sodium phosphate buffer) @ 4

 degree until the tissue has sunk down (a proximally over the day)

- Infiltrate the tissue with 20% sucrose (in sodium phosphate buffer) @ 4

 degree o/n until the tissue has sunk down

- Embed the tissue in Tissue – Tek O.C.T compound - Freeze in dry ice

b) Antibody

b.1) Antibody staining

Material

- PBS - TBS – Tween (0.3% tween) for 1 l: 10x TBS - weight 24.2g tris- base - weight 90g NaCl (sodium chloride) - add up to 990 mls of diH2O - mix - add 10 mls of concentrated HCL (12-13M) - pH 8

1x TBS - 100 mls of 10x TBS - 15 mls Tween20 - up to 1 l with water - mix

- TBS – Tween + 10 % goat serum - Primary antibody - Secondary antibody that correlate to the host animal from the primary

 antibody (f.ex.:  is the primary antibody produced in mouse than use an anti- 
 mouse antibody (cy5, cy 3 or other)

- Hoechst - Mounting media (90% glycerol in 0.02M Tris-CL pH 8.0 (stock solution:

 1M))

- Nail polish - Cover slips

Procedure

- Cut the sections on the cryostat (12-16 µm)

- Let dry @ Rt for 2 hours

- Wax around the tissue with DAKO pen

- Work in a humidity chamber

- Wash 3 times with TBS-Tween (0.3%) for 5-10 min each

- Block the sections with TBS-Tween+10% goat serum for 1 hour @ RT or o/n

 @ 4 degree

- Thaw the 1st AB (f.ex.: mouse anti- Pax7 – Hybridoma supernatants) @Rt or

 37 degree

- Spin the tube @13K for 10 @ 4 degree

- Add 100 – 200µl (in depend on the area which should be covered by the AB)

 to each slide

- Leave for few hours @ Rt or o/n @ 4 degree

- Wash with TBS-Tween 5 times for 10 min @ Rt

- Dilute the 2nd AB (f.ex.: anti- mouse Cy5 – should be stay in dark (alufoil) - )

 1:200 in TBS-Tween+10% goat serum

- Apply 100 – 150 µl of the AB and leave it for 2 hours @ Rt

- Wash 5 times with TBS-Tween

- Add Hoechst (final concentration 1µg/ ml; stock: 1 mg/ml – diluted in TBS-

 Tween) for 5 min 

- Wash once with TBS – Tween

- Mount and cover

Hybridoma cells

High serum media for the most monoclonal cell lines:

FBS (normal or depleted) 80 ml L – Glutamine (100x) 4 ml Gentamicin (100x) 4 ml DMEM fill up to 400 ml w/o Na pyruvate, with 4,5g/l glucose, with Piridoxine HCL

for cloning: use Hybridoma cloning factor (HCF) (Hybrid – max Hybridoma media supplement #ECO1021N (50 ml)) use @ 10 %

high serum media for Pax7

FBS (normal or depleted) 80 ml L – Glutamine (100X) 4 ml Pen/ Strep (100x) 4 ml Iscove’s DMEM fill up to 400 ml

in generell: 1) cells are NOT attaching on the bottom of the cell culture flask there are swimming in the media 2) for passaging they should not be detached by Trypsin 3) the cells need to be split 1:5 on Monday and Wednesday and 1:10 on Friday 4) Pax 7 should be grown up in 10% Co2 @ 37 degree, the other one in 5% CO2 @ 37 degree

Thawing of Hybridoma cells

1) thaw a vial of cells quickly @ 37 degree 2) transfer into 10 ml fresh High serum media 3) centrifuge for 3 min @ 1000 rpm 4) remove the supernatant 5) resuspent the pellet into 1 ml HS-media 6) transfer the cells into a well of a 24 well plate 7) take 0.5 ml of cells from the first well into the second well containing 1 ml HS-media 8) take 0.5 ml of cells from the second well into the third well containing 1 ml HS-media 9) take 0.5 ml of cells from the third well into the fourth well containing 1 ml HS-media 10) take 0.5 ml of cells from the forth well and put it back into the first well 11) incubate into the humidified incubator @ 37 degree by 5% (10%) CO2

When splitting the cells at each passage, gradually decrease the HCF concentration from 10% to 5%, 2,5% and 0 (if the cells are already stable, than you can start to grow up the cells also in HS – media w/o HCF). Check every time whether the cells have a good shape and looks healthy. In this way you have resuspended cells into 4 different concentrations. Than you should go up to work in 6 well plates and at the end in a 25 cm2 flask from corning.

Passaging of Hybridoma cells

Material

- HS- media - 15 ml falcon tube - 25 cm2 corning cell culture flasks (Pax 7 cells need the

Procedure

1) transfer the media including the cells into the falcon tube 2) centrifuge 3 min @100 rpm 3) remove the supernatant 4) resuspend the pellet into 5 ml fresh media and split 1:5 or 1:10 into new flask including 10 ml fresh HS-media

Freezing Hybridoma cells

Aliquotes of cells should be made and frozen as backup as soon as possible after defrosting.

Material

- Freezing media on ice (90% FCS, 10% DMEM) - falcon tube - freezing vials

Procedure

1) transfer the media including the cells into the falcon tube 2) centrifuge 3 min @100 rpm 3) remove the supernatant 4) resuspent the pellet (around 107 cells/ ml) in 1 ml freezing media 5) leave the cells for 24 hours at -80 degree and than transfer into liquid nitrogen

preparation of ‘depleted’ serum

Material

20 mM Na Phosphate buffer pH 7,5 500 ml HiTrap protein G column 1 ml 0.1M glycine pH 2,7 20 ml 1M Tris pH 9,0 10 ml FCS 100 ml PBS (10x) 10 ml Tubing Peristaltic pump Beakers

Procedure

1) thaw 100 ml FCS 2) add 10x PBS 1/10th of the volume of FCS 3) start pumping sterile H2O through the HiTrap protein G column at 1ml/min → at least 5 ml 4) when finished stop the pump (in the tubing shouldn’t come air!!!), change into the 20 mM Na Phosphate buffer pH 7,5 and start to pump at least 15 ml 5) put inlet of column at bottom of the flask containing the FCS, put outlet of the column (HiTrap) just above the FCS solution (in the same bottle) and run the FCS through 1 ml/ 1 min → circulate for 1 – 1.5 hours (on ice) or o/n @ 4 degree 6) save the “depleted” serum 7) sterile filter through 0,22 µm filter 8) freeze in aliquots 9) be careful!! → now it is 50% serum!!!!

To check the amount of IgG which binds to the column and to check if all IgG was removed from the FCS and to regenerate the HiTrap column:

a) prepare 10 eppendorf tubes for collecting the IgG which sticks to the column in fractions → label with 1 – 10 b) add into each tube 100 µl of 1M Tris pH 9.0 c) take 18 measurement cuvettes (8 for the standard curve and 10 for the fractions) d) thaw a IgG standard ( 1mg/ml) e) prepare the BIORAD reagent solution → dilute the stock 1: 5 (total volume at the end: 25 ml) f) prepare 20% EtOH (500 ml) g) after circulation with about 15 ml Na Phosphate buffer put the inlet of column in a tube with about 25 ml 0.1M glycine pH 2.7 h) turn on the pump and collect 1 ml of eluated AB in the eppendorf tubes containing 100µl 1M Tris pH 9.0 i) wash the HiTrap column by circulation with about 10 ml of 20 mM Na Phosphate buffer j) to store the column: circulate about 10 ml 20% EtOH through column IMPORTANT: for long term storage: in a “humified” environment (the best way would be to store the closed column in a 50 ml FALCON tube containing some drops 20% EtOH)

Check the concentration of the IgG into each fraction:

a) prepare the standard curve: cuvette µl of standard IgG (1mg/ml)

1 → 0 (zero point) 2 → 1 3 → 3 4 → 9 5 → 12 6 → 15 7 → 20 8 → 50

b) 10 µl of each fraction into the other 10 cuvettes c) add into each cuvette 1 ml BIORAD solution mix d) set up a spectrophotometer to Absorbance and OD 595 e) measure first the standards and than the samples f) prepare with the measurements of the standard curve and combine the measurements with this curve to figure out the concentration of each fraction → the last fractions should have the same absorbance like the zero point