Bioware® Brite Cell Culture Guidelines
Caution: For Research Use. This product is intended for animal research only and not for use in humans. Not for human or animal
therapeutic or diagnostic use.

STOPPerkinElmer recommends customers thoroughly read Cell Culture Guidelines aswell as product specific Technical Data sheet before working with cell lines.
As a courtesy, we have provided a second vial to be kept frozen as a backup.
If there is an issue after use of the first vial, DO NOT THAW second vial;
please contact PerkinElmer Technical Support Team at (800) 762-4000 or at
global.techsupport@perkinelmer.com

Important Notes
We strongly recommend Hyclone Fetal Bovine Serum (GE Healthcare Cat. No. SH300071)•

Please thaw only one vial for use to prepare your working stock. Freeze a backup stock of additional vials fromthe first few passage(s).

•Please see the specified growth medium composition as shown in Table 1 for each cell line.

•Each cell line grows at a different rate. Please refer to average doubling times in Table 1, and set culture
conditions and expectations accordingly.

•Previous guides may have recommended heat inactivation of serum. Please note that heat inactivation is not
required to achieve optimal growth of these cell lines.

•Do not use ANY antibiotic with GFP-expressing cell lines BW128090 and BW133416 as they do not have ANY
antibiotic selection resistance.

•Successive passages of non-GFP expressing cell lines can be achieved with or without antibiotic. Antibiotic in themedium is not required for optimal performance of the cells. However, it is recommended to add antibiotic if
potential bacterial contamination while growing cells is a concern in your lab. If antibiotic is to be added, please
note that only Puromycin at the correct final concentration should be added (as noted on TDS). Do not use anyother antibiotic.

•Please note that only % cell density and % confluence but not % viability can be determined by microscopic
visualization of cell lines. A viable cell count must be performed for all cell lines to assess true viability.

•BW124735 is a suspension cell line and does not require trypsinization for passage. When cells reach 80-90%
cell density/confluency in suspension, depending on doubling time obtained, simply dilute the culture 1:2 to 1:10
by plating in a bigger vessel containing fresh, warm media.

•BW124317 and BW119267 is a mixture of adherent and suspended cells. When % suspended celldensity/confluency is high and the plate looks full of cells, collect the culture media first to obtain the suspension
cells. If adherent cells are loosely attached, directly add trypsin without rinsing with PBS as cells maybe lost with
the PBS rinse. If adherent cells are tightly attached, a quick rinse with PBS followed by trypsin treatment isrecommended. Neutralize with 2x media. Pool all cells together, do a cell count and proceed to plate in a bigger
vessel with additional fresh media.

BW-Guidelines-Rev-10

Table 1.

ProductProduct Description

Media Composition*

Average Doubling Time(DT)***

BW124087

Bioware Brite 4T1-Red-FLuc

RPMI+10% Hyclone FBS

14BW128090

Bioware Brite 4T1-Red-FLuc-GFP**RPMI+10% Hyclone FBS

14BW124734

Bioware Brite B16F10-Red-FLuc

RPMI+10% Hyclone FBS

15BW128444

Bioware Brite PC3-Red-FLuc

EMEM+10% Hyclone FBS

24BW133416

Bioware Brite PC3-Red-FLuc-GFP**EMEM+10% Hyclone FBS

24BW124316

Bioware Brite NCI-H460-Red-FLuc

RPMI+10% Hyclone FBS

16BW125055

Bioware Brite LNCaP-Red-FLuc

RPMI+10% Hyclone FBS

60

BW134280

Bioware Brite HepG2-Red-FLuc

EMEM+10% Hyclone FBS

30BW124577

Bioware Brite U87MG-Red-FLuc

EMEM+10% Hyclone FBS

34

BW134246

Bioware Brite GL261-Red-FLuc

DMEM+10% Hyclone FBS

26BW128092

Bioware Brite HT1080-Red-FLuc

EMEM+10% Hyclone FBS

22BW125058

Bioware Brite BxPC3-Red-FLuc

RPMI+10% Hyclone FBS

36BW124353

Bioware Brite HT-29-Red-FLuc

McCoy’s 5a +10% Hyclone FBS

24BW124318

Bioware Brite HCT-116-Red-FLuc

McCoy’s 5a +10% Hyclone FBS

16BW124735

Bioware Brite K562-Red-FLuc****

RPMI+10% Hyclone FBS

15BW124317

Bioware Brite Colo205-Red-FLuc****

RPMI+10% Hyclone FBS

28BW119262

Bioware Brite MCF7-Red-FLuc

EMEM+10% Hyclone FBS

40BW119267

Bioware Brite LL/2-Red-FLuc****

DMEM+10% Hyclone FBS

24BW119276

Bioware Brite SKOV3-Red-FLuc

McCoy’s 5a +10% Hyclone FBS

35BW119266

Bioware Brite A549-Red-FLuc

RPMI+10% Hyclone FBS

22* Optional: Puromycin at a final concentration of 2 ug/mL for all cell lines listed above except for BW124087 which is at 5ug/mL.
** GFP cell lines (BW128090 and BW133416 do not have any antibiotic selection resistance).
*** Doubling time is an average. Actual doubling times will vary based on culture conditions and handling.

**** Suspension cell lines

BW-Guide-Rev-10

Thawing a Frozen Cell Vial

1. Thaw the vial rapidly by gentle shaking in 37°C water bath by hand. Be careful to keep the cap out of the water.
Wipe vial dry.
2. Spray the vial and your gloved hands with disinfectant (70% isopropyl alcohol) and wipe dry. Immediately after,
open the vial in the hood and transfer contents to 4mL of warm, sterile growth media with serum but noantibiotics. Mix gently. DO NOT CENTRIFUGE.
3. Count 1ml of the total cells and immediately plate the remaining cell suspension into a T25 flask. Incubate at
37°C, 5-6% CO2, 100% humidity overnight.
4. Next day, examine the cells under the microscope. If the cells are confluent, continue to instructions below for
passaging cell lines.
If the cells are not confluent:a. Aseptically remove the media and replace with 5mL of the same media warmed to 25ºC-37ºC.
b. Continue to incubate the plate(s) for an additional 1-7 days with minimal disturbance. Change
media every 3-4 days until the cells reach 80-90% confluency; only then proceed to passage thecells.
Passaging Cell Lines
1. For in vivo use we recommend less than 10 in vitro passages from original vial. However, split cells at least
one time before injecting in vivo.
2. When cells are approximately 80-90% confluent, passage cells to vessels with a 1:3 to 1:4 split without
antibiotic medium.
3. To passage the cells, remove media and add 5ml of sterile, room temperature 1X PBS. Gently swirl theadded PBS once over the cells and remove the PBS immediately.
4. Next, Add 1ml of 0.05% sterile, warm Trypsin (approximately 1mL for T25; 2ml for T75; 4ml for T150 and 5ml
for T175) to the flask containing cells and gently swirl to allow trypsin to coat the plate. Incubate at 37°C for 15 mins to allow cells to dissociate from the plate.
5. Examine the flask under a microscope to confirm dissociation. Neutralize with 2x medium, and gently
re-suspend the cells by pipetting up and down 1-2 times.
6. Transfer cells into a bigger flask (T75, T150, T175) at a 1:3-1:7 surface area ratio. Continue to incubate theplate(s) for 1-7 days with minimal disturbance. Change media every 3-4 days until the cells reach 80-90%
confluency; only then proceed to passage the cells.

Creating Cell Stocks
1. When cells have reached 80% confluence, freeze aliquots for 24 hours in -80ºC in 5% DMSO/95% FBS without
antibiotics. Transfer frozen vials to LN2 tank after 24 hours.
2.

We recommend that you thaw one test vial to check and confirm viability by cell counting and/or culturing.

BW-Guide-Rev-10