Transfection of HEK293TLD in suspension using PEI Max
PROTOCOL (modified from Leila’s)
CELL LINE MAINTENANCE
Stock: >20 million cells per vial (~1.8ml) (FBS with 10% DMSO: 9ml of FBS + 1ml of DMSO)
Starting culture: 1 vial of stock (20 million cells) in 50mL of media
Cells are generally maintained at a density of -0.2 to 4 million /ml. Cells will grow to 7 million/ml but the growth slows after ~5 million/ml. Average double time is roughly 36 hours.
To make stock, add Blasticidin (@ 10ug/ml final concentration) to culture media, let the cells double at least twice (Blasticidin can be stable in media for 2 weeks). Harvest cells by centrifugation (1000 g for 10’ @ 4C). Add FBS with 10% DMSO to the cells (should have > 10 million cells per ml), aliquot ~1.8ml in each vial.
FreeStyle™ 293 Expression Medium (ThermoFisher, Catalog number: 12338018) with 1% Tet free FBS, 2mM L-Glutamine (ThermoFisher, Catalog number: 25030-081, 200mM, 100x) and Phenol red (Sigma P0290, 0.5%, liquid; final concentration 0.015g/L)
CELL COUNTING
From Taylor et al 2016: “HEK-293Ts grow in small clumps of 1-30 cells in suspension. Accurate counting requires dissociation of the clumps by gentle tituration using a pipet. Due to differences in light scattering by different clump sizes, optical density is not an accurate measure of cell number. We visualize the cells before and after dissociation because shearing in the dissociation protocol lyses a small fraction of the cells.
1. Using a 1mL serological pipet, aliquot 200uL …of culture to a clean microcentrifuge tube.
2. Mix by flicking, and pipet 10uL onto one side of the hemacytomer.
3. With a 200uL pipet, set the volume to ~180uL and titurate 30x to break up the clumps. Try not to foam.
4. Pipet 10uL onto the remaining half of the hemocytometer”
TRANSFECTION
Transfection is done using 1ug DNA/mL total culture volume. Transfection reaction is performed in 1/20th of the culture volume, with a 3:1 ratio of PEI Max to DNA.
Prep DNA: DNA should be prepared fresh via endotoxin-free midi/maxi prep. Use 1mg of DNA per 1L of culture
DAY ONE (Base on 1L of culture volume)
1. Grow cells to 2.0-4.0 million/mL.
2. Count cells and record numbers.
3. Warm 50mL hybridoma medium (or any suitable serum-free medium) to RT
4. Dilute 1000ug DNA in the hybridoma media, mix well
5. Add 3000uL 1mg/mL PEI Max (pH 7.0) Mix well
6. Incubate for 15 minutes at RT to allow DNA-PEI complex to form (not longer!)
7. Add to culture flasks with serological pipet and return to incubator
DAY TWO
8. Count cells. Induction can be done on this day or DAY THREE. Induction is done by adding 1ug/mL doxycycline to the cultures. (Doxycycline stock 10 mg/ml, 10,000x)
DAY THREE
9. Count cells. Induce as above, or Harvest cells by centrifugation.
DAY FOUR
10. Harvest cells by centrifugation (1000 g for 10 min @ 4C)
EXPERIMENTAL PROCEDURE
Transfection culture
8/11/21
Thaw 1 vial of stock and inoculate in 30ml of media
8/12/21
~270K/ml (8/13/21, same number of cells)
8/16/21
~1million/ml (1M/ml); add 50ml media
8/17/21
~370K/ml
8/20/21
3 bottles for transfection: 1 bottle of 100ml culture @ 2M/ml; 1 bottle of 50ml culture @ 2M/ml Dilute initial culture, make 3 flasks of 200ml culture, ~0.5million/ml for transfection and induction
1 bottle of 50ml culture, ~1M/ml for stock
Add 100ml of media (total volume 150ml)
Add 150ul of blasticidin 10mg/ml stock (1000x), final concentration 10ug/ul
8/23/21
Transfection culture: ~2M/ml; add 150ml to each culture
8/24/21
Count cells of cultures for transfection ~3M/ml
Preparation for transfection (calculation is based on 1L culture)
Total 1100ml of culture (350 + 350 + 400)
1) Warm up 55ml of FreeStyle medium without FBS
2) Add 1.1mg of DNA to the medium (2220ul total) to the medium, mix well
3) Add 3300ul 1mg/ml PEI Max, pH7.0, mix well
4) Incubate for 15 minutes at RT to allow DNA-PEI complex to form (not longer! No need to shake)
5) Add to culture flasks with serological pipet and return to incubator
Total volume of the transfection mix: 55ml medium + 2.22ml DNA + 3.3 ml PEI Max ->60.5ml Add 19.25ml to each of the two 350ml culture and 22ml to one 400ml culture
8/25/21
Count cells or transfection cultures (3 flasks ongoing)
All fresh medium to #1 (100ml), #3 (150ml) and # 4 (100ml), so all flasks will have 350ml of culture and @ ~2M/L concentration
Add 35ul of 10mg/ml Doxycycline stock to each flask (final concentration will be 1ug/ml)
Induce @ 1pm (1ug/ml Dox)
8/26/21
Harvest cells @ 1pm (24h induction); 1000g for 5’ @ 4C
Wash twice with cold 1x PBS, 1000g for 5’ @ 4C
Make BB’s and grind (total 13g of powder from 1.4 L of culture)
Testing new MT302 powder
August 27, 2021 - Testing new MT302 powder
Maintenance culture for making stocks
8/20/21
1 bottle of 50ml culture, ~1M/ml for stock
Add 100ml of media (total volume 150ml)
Add 150ul of blasticidin 10mg/ml stock (1000x), final concentration 10ug/ul
8/23/21
Stock culture: 0.5M/ml, add 50ml medium to 150ml culture (since the color of the medium already turned a little bit orange)
8/24/21
Count cells: still @ 0.5 M/ml (100 million cells total in 200ml culture)
Make 10ml FBS/10% DMSO (9ml of FBS + 1ml DMSO)
Calculate total number of cells
Spin down the culture 1000g, 10’ @ 4C
Resuspend cells in 5.4 ml of FBS/10% (5.4 mls, make 3 vials of stock; ~33 million in each vial) Aliquot 1.8ml (>20million cells) into cryovials
Keep the stock in Styrofoam container @ -80C for few days and then transfer to liquid nitrogen
Start another maintenance culture to make more stock
50ml of culture @ 2M/ml
Add 100ml of media (total volume 150ml)
Add 150ul of blasticidin 10mg/ml stock (1000x), final concentration 10ug/ul
8/27/21
Count cells of the culture for making stocks
150ml @ 1.2 M/L, 150 million cells total, enough to make 7 vials of stocks; each vial will have >20 million cells (25M)
Made another 7 vials of stock (same procedure as described above)
8/30/21
Put all 10 vials of stock in liquid N2 tank (Box “Mehrnoosh”)