Jun 16, 2025
Generating ZsGreen+/- OVA Cell Lines
This protocol is a draft, published without a DOI.
- Kasidy Brown1
- 1OHSU
Protocol Citation: Kasidy Brown 2025. Generating ZsGreen+/- OVA Cell Lines. protocols.io https://protocols.io/view/generating-zsgreen-ova-cell-lines-g3b7byirp
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 14, 2025
Last Modified: June 16, 2025
Protocol Integer ID: 220255
Keywords: new cell line, identification of transfected cell, transfected cell, visualization of antigen uptake, cell, zsgreen, ova, observing antigen, line, specific responses via ot, ii assay, antigen uptake
Abstract
To create a new cell line that expresses ZsGreen and/or OVA to allow visualization of antigen uptake, identification of transfected cells, or for observing antigen-specific responses via OT-I/OT-II assays.
Materials
1. CalPhos Mammalian Transfection Kit (Cat #: 631312)
a. 9mL 2M Calcium Solution
b. 2 x 35mL 2x HEPES-Buffered Saline (HBS) - Keep at -20°C
c. 2 x 35mL Sterile H2O
2. 6-well TC Treated Plates
3. MB49 Line (or cell line of choice)
a. 50-80% confluent on day of use
b. *Cells need to be plated the day before use in protocol*
4. 12 x 75-mm sterile tubes
5. D12 Media (500mL) (or media of cells)
a. 10% FBS
b. 1% P/S/G
6. PBS
a. pH = 7.4
7. 1x Trypsin/EDTA
8. Plasmid DNA
| A | B | C | D | |
| Plasmid | Section in -20 | Concentration (ug/uL) | Volume (uL) | |
| pSiren ZsGreen | Row 4, Slot 28 | 0.239 | 400 | |
| pCAGs ZsGminOVA | Row 5, Slot 37 | 1.097 | 400 |
This will be adjusted according to what you plan to transfect with
Troubleshooting
Culturing MB49 (use proper thaw technique for cell line you plan to transfect)
Thaw cell line by placing cell vial in 37°C bath just until it thaws
Place 1mL cells in 10mL D10-MB49 (D12) media
Spin down cells at 1500 rpm for 5 min
Aspirate supernatant and resuspend in 5mL D12 media
Plate cells in T150 flask with 20mL media
Split cells as needed, performing a 1:20/1:30 split as cells grow rapidly
Should split at least twice before performing transfection to ensure cell lines come out of thaw properly
Plating MB49 Cells for Transfection: Day 1
Split cells 1:20 so that cells are ~50-80% confluent the next day
Plate cells in 2mL in 6-well TC-treated plates
Two 6-well plates, 3 wells for each condition to optimize cell density
- do this when performing transfection for the first time- helps with determining which cell density generates the highest reporter gene activity
Well 1: 5x104
Well 2: 2x105
Well 3: 4x105
CalPhos Mammalian Transfection Protocol: Day 3 (11/17/2024)
For each transfection, prepare solution A and Solution B in separate tubes
Solution A.1: ZsGminOVA
- use table to calculate how many uL of plasmid is needed to add final concentration of 2ug DNA to wells.
- Total volume = 100uL for one well, calculate amount of H20 needed to bring volume up to 100uL
Example: ZsGminOVA is at 1ug/uL
| A | B | |
| 2uL | 2ug plasmid DNA needed | |
| 85.6uL | Sterile H20 | |
| 12.4uL | 2M Calcium Solution | |
| 100uL | Total Volume |
Solution A.2: ZsGreen
- use table to calculate how many uL of plasmid is needed to add final concentration of 2ug DNA to wells.
- Total volume = 100uL for one well, calculate amount of H20 needed to bring volume up to 100uL
Example: ZsGreen is at 0.25ug/uL
| A | B | |
| 8uL | 2ug plasmid DNA needed | |
| 79.6uL | Sterile H20 | |
| 12.4uL | 2M Calcium Solution | |
| 100uL | Total Volume |
Solution B: 100uL 2X HBS
Calculate master solution for amount of transfections
- Example: I have two wells from each condition that will be transfected, therefore I need 200uL total volume for each solution A and 200uL for each solution B
Solution A.1: ZsGreenminOVA
| A | B | C | |
| 2uL | 2ug plasmid DNA needed | need 4uL | |
| 85.6uL | Sterile H20 | need 171.2uL | |
| 12.4uL | 2M Calcium Solution | need 24.8uL | |
| 100uL | Total Volume | 200uL |
Solution B.1: 200uL 2X HBS
Solution A.2: ZsGreen
| A | B | C | |
| 8uL | 2ug plasmid DNA needed | need 16uL | |
| 79.6uL | Sterile H20 | need 159.2uL | |
| 12.4uL | 2M Calcium Solution | need 24.8uL | |
| 100uL | Total Volume | 200uL |
Solution B.2: 200uL 2XHBS
Carefully and slowly vortex Solution A while adding Solution B dropwise
Incubate the transfection solution at RT for 10min (5-15min)
Gently vortex transfection solution and then add solution dropwise to culture plate medium. (Add 200 μl of transfection solution per well of a 6-well plate.)
Gently move plates back and forth to distribute transfection solution evenly. (Do not rotate plates as this will concentrate transfection precipitate in the center of the well or plate.)
Incubate plates at 37°C for 8 hr–overnight in a CO2 incubator.
Washing and Feeding Transfected Cells: Day 4
Remove calcium phosphate-containing medium and wash cells with medium or 1X PBS.
Feed plate with 2 ml fresh complete growth medium and incubate at 37°C until needed for assay.
Wells may need to be split by end of day
Move all contents into T150 and bring up to 25mL with media
Assay for transient gene expression or start selection for stable transformants 24–72 hr post-transfection.
Sort #1: Day 6
Harvest transfected cells around 80-90% confluency
Pool densities together
Harvest non-transfected MB49 for non-transfected control
- either have non-transfected cells growing in a flask or you can thaw frozen cells from stocks
Spin down cells at 1500rpm for 5 minutes
Bring up each pellet in 1mL DAPI
Sort cells into 15mL conicals with 7mL MB49 media
Sort MB49 that successfully took up plasmid (will be ZsGreen+)
Record how many cells were sorted from each group and what percent were positive for ZsGreen
- Example: Sorted 15k cells for MB49ZsGHi, 15k cells from MB49ZsGminOVA
- got 0.01% positive for ZsGreen in both conditions
Plate cell strains in a 24-well plate with 1mL D12 media
- Plating and media will vary based on how many cells are collected. Start with smaller culture conditions when plating smaller volumes of cells; cells like to be close by one another
Splitting Cells: Day 8, 10, 12, 14, etc. (perform at least 2 splits to ensure stable integration of construct)
When cells are at 80% confluency, split at 1:20 ratio so cells can be harvested every other day
- record the days/how many times splits were performed
__/__/__: Split cells
__/__/__: Split cells
__/__/__: Split cells
Sort #2: Day 26 (this will vary)
Harvest transfected cells (MB498ZsG- % confluent; MB49ZsGminOVA- % confluent)
- record %confluency on day of sort
Harvest non-transfected MB49 for control
Spin down cells at 1500rpm for 5 minutes
Bring up each pellet in 1mL DAPI and transfer to a 5 mL polystyrene round bottom tube
Sort cells into 15mL conicals with 7mL MB49 media
Only collecting MB49ZsGHi and MB49ZsGminOVA; not MB49 control
Sort MB49 that successfully took up plasmid (will be ZsGreen+)
- record number of cells sorted and %ZsGreen+
- Example: sorted 15k cells MB49ZsGreenHi (0.6% positive), sorted 200k cells fro MB49ZsGreenminOVA (10% positive)
Plate cells according to number collected
- Use corning guide to determine how many cells should be plated in which type of plate and how much media to use
Sort #3: Day 35 (this will vary)
Harvest transfected cells, record %confluency
- Example: MB49ZsG- 75% confluent; MB49ZsGminOVA- 50-60% confluent
Harvest non-transfected MB49 for control
Spin down cells at 1500rpm for 5 minutes
Bring up each pellet in 1mL DAPI and transfer to a 5 mL polystyrene round bottom tube
Sort cells into 15mL conicals with 7mL MB49 media
Sort MB49 that successfully took up plasmid (will be ZsGreen+)
- record amount of cells collected and %ZsGreen+
Record cell density and plating conditions
Sort #3: Day 42 (this will vary)
Harvest transfected cells, record %confluency
Harvest non-transfected MB49 for control
Spin down cells at 1500rpm for 5 minutes
Bring up each pellet in 4mL MACs and transfer to a 5 mL polystyrene round bottom tube
Sort cells into 15mL conical with 7mL MB49 media
Sort MB49 that successfully took up plasmid (will be ZsGreen+)
- record amount of cells collected and %ZsGreen+
Record cell density and plating conditions OR if finished sorting, freeze down all cells leaving a flask growing to make sure freeze goes well
- perform test thaw before you stop culturing newly transfected line