• Cell culture splitting
• Flow cytometry
• Gibson assembly
• Immunofluorescence labeling
• Transformation of E. coli
• Two step PCR

• Maxiprep
• Miniprep

• Electroporation of COS Cells
• Transfection of HEK293T cells with Metafectene
• Transfection of ExpiCHO
• Transfection of Expi293

Preparation for Maxiprep: Pick clones and inoculate culture

Preparation for Maxiprep: Centrifuge cells
 → only with 0_TEV, M_tTA (have grown o/n, G_tTA has not)
 → Preparation for Maxiprep: pick clones and inoculate culture (G_tTA)

Preparation for Maxiprep: Centrifuge cells (G_tTA)

Maxiprep: M_tTa and 0_TEV cells frozen o/n, G_tTA cells centrifuged right before
 → yield of G_tTA really low (500 µL with 0.48 µg/µl), fine for now but backup cells would be good
 → Preparation for Maxiprep: pick clones and inoculate culture (G_tTA)

Preparation for Maxiprep: Centrifuge cells (G_tTA)

Preparation for Maxiprep: centrifuge cells (G_tTA)
 → x2
 → stored in -20 °C freezer ready for Maxiprep when needed

Splitting COS cells

Preparation for Maxiprep: pick clones and innoculate culture (eYFP, Tev, tTA_BFP)
 → picked from stab culture into 5 mL LB + 1x Ampicillin, incubated at 37 °C for ~4 h
 → transferred to 0.5 L LB + Amp, incubated overnight

Maxiprep (eYFP, TEV, tTA_BFP):
 → eYFP: 4 µg/µL
 → TEV: 2.56 µg/µL
 → tTA_BFP: 2.58 µg/µL

Splitting COS for Electroporation

Electroporation with dTomato MESA + Reporter:
 → concentrations as described
 → 3 µL of 0.5 µg/µL DNA added

Next time: empty plasmid to even out DNA amount/ plasmid count?

Splitting CHO-cells: for Transfection on 24.08.2022
 → 80 mL in 250 mL flask
 → 5.33 x 10⁶ cells/mL 99% (5.04 x 10⁶ cells/mL 99% & 5.62 x 10⁶ cells/mL 99%)
 → 8.88 mL cells and 71.12 mL media

Trafo

Inoculate cultures: with GFP-GFP, mCherry-mCherry, GFP-mCherry, GBP, MBP

D-1 Splitting cells: to density of 4 x 10⁶ cells/mL for 75 mL in 250 mL flask

Maxiprep: with GFP-GFP, mCherry-mCherry, GFP-mCherry, GBP, MBP

Cloning:
 → Digest GBP & MBP Scheller plasmids with EcoRI, XbaI
 → PCR on V2-MESA tTA and V2-MeSA TEV for linker 1 and linker 2 variants
 → 4 samples total: 0.1 µg plasmid, 58 °C annealing, 4min elongation
 → gel purification of digest & PCR
 → Digest purified PCR EcoRI, XbaI
 → PCR purification
 → Ligation 15 min at RT 50 ng vector, 49 ng tTA or 22.8 ng TEV
 → 4 samples
 → WRONG! do ligation overnight next time (wrong protocol)

Trafo: of 5 µL ligation

Transfection: of ExpiCHO with mCherry Fc, GFP mCherry, and GFP Fc
 → 1.165 x 10⁷ cells/mL 99% (1.33 x 10⁷ cells/mL 99% & 1.00 x 10⁷ cells/mL 99%)
 → 12.88 mL cells and 12.12 mL media (for 25 mL Transfection with cell density of 6 x 10⁶ cells/mL in 125 mL flask)

Plates don't look good
 → dephosphorylate remaining vector
 → ligate all 8 samples overnight at 4 °C + neg control (only vector)

Ligation: G1TA & M1TA again

Miniprep: G2TA, M2TA, M2TEV → sequencing

Inoculate clones 2(G2TEV), 9(M2TEV), 10(M2TA), 17(M1TEV) for maxi tomorrow

Repick G2TA, G1TEV 5x

Prepare protein A buffers, set pH at 4 °C!

Protein a purification of ligand-Fc samples:
 → Filter samples before FPLC (25 mL per sample)

• wash 3CV buffer A
• load
• 5-10CV A (collect)
• 6CV B (collect + neutralize 1/10 of Volume with Buffer N) → add beforehand to tubes
• regenerate 5cV B
• wash 5CV A

 → GFP-Fc: A13-A18
 → GFP-mCherry: A13-18
 → mCherry: A41-A45
 → + 1x load & 1x flowthrough for SDS gel
 → run gels (see pictures)

• GFP-Fc: A13-A18
• GFP-mCherry: A14-18
• mCherry: A41-A45

• GFP-Fc: 194 µg/mL
• GFP-mCherry: 117 µg/mL
• mCherry: 100 µg/mL

Miniprep: G1TA, M1TA → sequencing

Electroporate 4 µg total:

See standard protocol

Immunostaining: 16B12 & 05-724 + mouse & rabbit secondary Ab

Electroporation for FACS:
 → 0.5 µg/µL DNA, maxiprepped
 → 3-5 x 10⁶ cells/mL, 200 µL
 → 3 x 10⁴ cells/mL in 2 mL in 6 well plate

FACS

Microscopy: electroporate 100 µL x 10⁶ cells/mL

Add Ligand (mCherry-Fc) to FACS samples (see list from yesterday)

mCherry-Fc Stock: 100 µg/mL
 → MESA + Ligand500: 10 µL in 2 mL media
 → MESA + Ligand100: 2 µL in 2 mL media

Change buffer for all wells

Pick more M1TA, G2TA

FACS measurments: transfection efficiency horrible (maybe solution too old)

Mini-prep + sequencing M1TA, G2TA

Electroporation test in 4 mm cuvettes (250 µL electroporation solution, 5 µg dna, 4.5 x 10⁵ cells/mL)

IF Staining of cells

Fluorescence microscopy: pretty much no difference between samples, better than before, but still pretty bad

Cloning: 2 x mCherry-t2A eYFP and GFP-mCherry-eYFP
 → digest eYFP reporter with NotI, HindIII
 → PCR: eYFP T2A, mCherry 1, mCherry 2, GFP-mCherry
 → 4 min elongation,  °C annealing
 → gel purify

Electroporation for FACS:
 → 0.5 µg/µL DNA, maxiprepped
 → 2 x 10⁶ cells/mL, 250 µL (4 mm cuvettes)
 → 2.5 x 10⁵ cells/mL in 2 mL in 6 well plate
 → 5 µg total DNA on cells

FACS

Pick some more M1TA

Gibson assembly: 2xmCherry-t2A eYFP and GFP-mcherry-eYFP
A. mCherry2x
 → 134.7 ng BB
 → 26.52 ng mCH 1
 → 22.81 ng mCH 2
 → 25.09 ng EYFP T2A
B. GFP-mCherry
 → 100 ng BB
 → 72.78 ng GFP-mCherry
 → 37.24 ng eYFP T2A

Add ligand mCherry 500 ng/mL

Pick GFP mCherry & 2xmCherry

Prep & sequence:
 → M1TA
 → GFP mCherry, 2xmCherry

Transfection: HEK293T with Metafectene as per protocol
 → 4 µg DNA total, 0.5 µg/µL plasmids

Digest: dTomato_TEV, eYFP → NotI, HindIII

Digest: GBP-gp130 → XbaI, AflII

Addition of 500 ng/mL ligand according to table above

Cytometry Measurements

Transfection: HEK293T with Metafectene as per protocol
 → 4 µg DNA total, 0.5 µg/µL plasmids

Add ligand according to table above

Cytometry Measurements

Redo PCR for 2xmCherry T2A EYFP and GfP-mCherry t2A eYFP
 → two temp annealing 10 cycles low, 23 high
 → gel purify, redo gibson like 20.9.

Maxiprep with miRFP reporter plasmid

Miniprep with MESA component plasmids G1TA G2TA und G1TEV

Transfection: HEK293T with Metafectene as per protocol
 → 4 µg DNA total, 0.5 µg/µL plasmids

Maxiprep with G1TA G2TA und G1TEV

Addition of ligand according to table above

Cytometry measurements

Splitting of ExpiCHO & Expi293

Preparation for Maxiprep: transformation of E.coli with pH plasmid

Splitting of ExpiCHO & Expi293

Preparation for Maxiprep: pick clones and inoculate culture

Preparation for Maxiprep: centrifuge cells, freeze for tomorrow

Maxiprep with pH plasmid

Transfection: of ExpiCHO & Expi293 with pH plasmid (T0)

Feeding ExpiCHO & Expi293 (T1)

Take sample of ExpiCHO & Expi293 (T2)
 → CHO: 93% live, 1.34 x 10⁷ cells/mL & 94% live, 1.35 x 10⁷ cells/mL
 → 293: 75% live, 5.33 x 10⁶ cells/mL & 77% live, 6.11 x 10⁶ cells/mL

Take sample of ExpiCHO & Expi293 (T3)
 → CHO: 90% live, 1.36 x 10⁷ cells/mL & 89% live, 1.35 x 10⁷ cells/mL
 → 293: 75% live, 5.33 x 10⁶ cells/mL & 77% live, 6.11 x 10⁶ cells/mL

Take sample of ExpiCHO & Expi293 (T4)
 → CHO: 94% live 1.32 x 10⁷ cells/mL & 94% live 1.27 x 10⁷ cells/mL
 → 293: 74% live 8.16 x 10⁶ cells/mL & 67% live 7.88 x 10⁶ cells/mL

Take sample of ExpiCHO & Expi293 (T7)
 → CHO: 91% live 1.57 x 10⁷ cells/mL & 91% live 1.17 x 10⁷ cells/mL
 → 293: 23% live 3.06 x 10⁶ cells/mL & 17% live 3.07 x 10⁶ cells/mL

Take sample of ExpiCHO & Expi293 (T8)
 → CHO: 92% live 1.33 x 10⁷ cells/mL & 89% live 9.65 x 10⁶ cells/mL
 → 293: 7% live 1.0 x 10⁶ cells/mL & 57% live 7.27 x 10⁵ cells/mL

Take sample of ExpiCHO & Expi293 (T9)
 → CHO: 90% live 1.23 x 10⁷ cells/mL & 88% live 1.09 x 10⁷ cells/mL
 → 293: 5% live 8.21 x 10⁵ cells/mL & 6% live 1.21 x 10⁶ cells/mL

Prepare SDS-Gels for Expression Analysis

Take sample of ExpiCHO & Expi293 (10)
 → CHO: 88% live 1.30 x 10⁷ cells/mL & 85% live 1.56 x 10⁷ cells/mL
 → 293: 2% live 4.57 x 10⁵ cells/mL & 2% live 4.05 x 10⁵ cells/mL

Take sample of ExpiCHO & Expi293 (11)
 → CHO: 84% live 7.68 x 10⁶ cells/mL & 82% live 8.13 x 10⁶ cells/mL
 → 293: 3% live 4.11 x 10⁵ cells/mL & 5% live 6.57 x 10⁵ cells/mL

Load SDS-Gels with Expression Samples
 → 100 µL mixed with 25 µL 5x reducing Lämmli
 → 4 Gels: 293: SDS & Western Blot (WB), CHO: SDS & WB
 → Samples: T2, 3, 4, 7, 8, 9, 10, 11 on each gel
 → 3.5 µL of marker (PageRuler Plus Prestained) "M"
 → Unused Wells filled with 1x Lämmli (1xL)

Gel 1: 293 SDS
(accidentally inverted sample application)

Gel 2: 293 WN

Gel 3: CHO SDS
(accidentally inverted sample application)

Gel 4: CHO WB

Run at 150V for 1.5 h

Transfection of ExpiCHO (D1)

Transfection of ExpiCHO (D0), pH linker

Feed CHO cells (D1)

Harvest cells (D6)
 → centrifuge at 4 °C in 6 x 50 mL Falcons 5000 x g, 30 min
 → take supernatant, centrifuge at 4 °C for 15 min 20000 x g

Dialysis o/n
 → Protein in dialysis tube, fixated with white clamps
 → in 5 L 20 mM Tris/HCl pH 7.5 at 4 °C

Prepare SDS-Gels: 12 % gels x4

Prepare IMAC
 → Binding buffer: 20 mM Tris/HCl, pH 7.5 at 4 °C (2 L prepared)
 → Elution buffer: 20 mM Tris/HCl, pH 7.5, 500 mM imidazole at 4 °C (500 mL prepared)

Prepare Gefi
 → 200 pg column
 → 20 mM Tris/HCl pH 7.5 100 mM NaCl

IMAC

SDS-PAGE
 → Fractiones from IMAC were selected with the chromatogramm (detection at 280 nm)
 → 40 µL samples + 10 µL 5x Laemmli + 10% β-Mercaptoethanol, heated at 95 °C
 → 3.5 µL of marker (PageRuler Plus Prestained) "M"

Concentrate Protein

Buffer preparation for pH-sensitivity experiment:
 → leave in Tris buffer for pH 7.5 for one sample (dilute as others)
 → pH 7, 6.8, 6.5, 6.3, 6: make 500 mL 100 mM NaH2PO4 + 100 mM NaCL, set pH using NaOH in 50 mL falcons
 → dilute pH protein in tube in 0.5 mL by factor 24 (to 0.2 µg/µL)
 → immediately take one sds sample from each tube (2 µg) add Lämmli, heat shock, freeze for tomorrow
 → leave samples overnight at 37 °C.

SDS Gel: from overnight sample, load 2 µg of protein from each sample