Editing Hybridoma production (section)

== Fusion Protocol ==

-Prewarm all mediums and PEG 1500 to 37°C.

'''Typically need:'''

1-1.5L HAT medium (assuming 3-5 x 10<sup>8</sup>&nbsp;splenocytes)

~100ml HYB-SFM + 10% FBS

Corning DMEM: 10-017-CV, with 4.5g/L glucose, L-glutamine without sodium pyruvate

[https://us.vwr.com/store/product/9302661/dulbecco-s-modification-of-eagle-s-medium-dmem-corning https://us.vwr.com/store/product/9302661/dulbecco-s-modification-of-eagle-s-medium-dmem-corning]

Hybridoma-SFM:&nbsp;Catalog number: 12045076,&nbsp;[https://www.thermofisher.com/order/catalog/product/12045076 https://www.thermofisher.com/order/catalog/product/12045076]

~25ml HYB-SFM, no FBS

Hyclone Alpha CALF Fraction: SH30212 03

Red Blood Cell Lysis Buffer Hybri-Max: Sigma R-7767, 100 ml

Amphotericin B solution: 0.25 mg/ml 03-028-1B, 100 ml, from&nbsp; &nbsp;Biological Industries

'''Home-made Amphotericin B''': 200,000X: weight ~ g in H2O, suspension,&nbsp;

Add 0.5 ml to 100ml 100X Penstrep., Add 10 ml or 5 ml of this 100X&nbsp; stock to 500 ml DMEM/FBS.



Gentamycin Sulfate: 50 mg/ml as base, 03-035-1B, 100 ml,&nbsp;from&nbsp; &nbsp;Biological Industries

L-Glutamine from Biowhittaker 200 mM solution, 100 ml

[https://www.fishersci.com/shop/products/lonza-biowhittaker-l-glutamine-200mm-l-glutamine-200mm-100ml/bw17605e https://www.fishersci.com/shop/products/lonza-biowhittaker-l-glutamine-200mm-l-glutamine-200mm-100ml/bw17605e]

Cell Strainer 100 um Sterile

Sigma Hybri-Max PEG solution, P7181, 5 ml,&nbsp;[https://www.sigmaaldrich.com/catalog/product/sigma/p7181 https://www.sigmaaldrich.com/catalog/product/sigma/p7181] Gerbu Adjuvant MM, #3001A, 6x1ml, Accurate Chemical & Scientific Corp.<br/>Hybridoma-SFM, #12045-076, 1000ml, Invitrogen<br/>HAT, H0262-10VL, 10 vials, Sigma<br/>HT, H0137-10VL, 10 vials, Sigma<br/>Recombinant human IL6, RIL61, 20ug, Endogen/Pierce<br/>PEG 1500, #783641, 10x4ml, Roche Diagnostics



'''Myeloma cells'''<br/>-Assess and count X63-Ag8.653 myeloma cells. Leave in Hyb+10%FCS in incubator throughout spleen cell recovery.<br/>(X63-Ag8.653s grow best in 150mm tissue culture treated petri dishes, approx. 60&nbsp;ml each. Aim for 8 plates per fusion. This usually yields twice as many cells as needed&nbsp;for the fusion.&nbsp;X63s are thawed the week before the fusion, and are split out 1:2 each day until&nbsp;the day before the fusion (i.e. Monday through Wednesday) to keep in exponential&nbsp;growth.)

'''SP2 cells:&nbsp;'''

Sp2/0-Ag14, $431,[https://www.atcc.org/en/Products/Culture_Reagents/Hybridoma_Development/CRL-1581.aspx https://www.atcc.org/en/Products/Culture_Reagents/Hybridoma_Development/CRL-1581.aspx]

Sp2/mIL-6,&nbsp;$551.00, &nbsp;[https://www.atcc.org/en/Products/Culture_Reagents/Hybridoma_Development/CRL-2016.aspx https://www.atcc.org/en/Products/Culture_Reagents/Hybridoma_Development/CRL-2016.aspx]

Culture medium for Sp2/0: DMEM+FBS 10%, 1% p/s medium

<br/>Spleen cell collection<br/>1. Euthanize mouse via cervical dislocation or CO2

2. Place entire mouse in a beaker containing ~200ml of 80%Betadine, 20% 70%-ethanol, or 70% ethanol

3. Remove spleen using aseptic techniques<br/>(use 3 sets of tools (scissors, forceps), one each for skin, IP sheath,&nbsp;spleen)

4. Transfer spleen to 50 ml sterile tube&nbsp;containing 5ml Hyb SFM + 10%FCS&nbsp;&nbsp;

5. In hood, move spleen to new Petri dish in 1ml Hyb-SFM+10%FCS.

6. Trim fat and connective tissue, then frush out spleen cells using neddle

or cut spleen into small pieces: cut spleen into 5 sections longitudinally, then cut each section in half

Using two sterile watchmaker’s forceps, secure a piece of spleen with one forceps while “milking” cells from the piece into the medium.

(Place capsule pieces aside in a pile in the petri dish.)

7. Sieve-tissue grinder: one glass tissue grinder, one Bellco&nbsp;Cellector Tissue Sieve Kit 85mL

grinder the leftover tissue using ethanol sterilzed&nbsp;Sieve-tissue grinder

rinse&nbsp;leftover tissue with serum-free DMEM medium&nbsp;

8. Filter through Cell Strainer into another 50 ml tube.

(Also wash capsule sections. Wash “main” spleen cell collection 4 or 5&nbsp;times, and capsule collection 2 or 3 times. Transfer all material, including&nbsp;chunks and capsules, to 50ml tube)

<br/>9.&nbsp; Centrifuge at&nbsp;900-1000rpm for 5min.

10. Pour off supernatant inside the&nbsp;hood,

11. Add 1.2 ml of Red Blood Lysis Solution

12. Incubate at RT for 1&nbsp;min

13. Add 25 ml of serum-free medium

14. Take 0.2 ml out for cell counting (Cellometer Mini, Nexcelom BIOSCIENCE )

(Don’t count obvious RBCs, but when in doubt, count)

15. Calculate required number of X63s or SP2 cells&nbsp; (i.e., 1:10 or 1:2 ratio, spleen cells:SP2 cells).&nbsp;

Rick's protocol: 1:10 ratio

16. Combine spleen cells and SP2 cells into one 50 ml tube

(if desired, place combined cells in 37C incubator and go have lunch)