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Rapid Development of Monoclonal Antibodies Using Repetitive Immunizations, Multiple Sites
|Rapid Development of Monoclonal
Antibodies Using Repetitive
Immunizations, Multiple Sites
An in-depth understanding of both the formation of
germinal centers and the immunoregulatory processes
involved in T-cell-dependent B-cell responses (Levy et al.
, 1989; Berek et al.
, 1991; Jacob et al.
, 1991; Kroese et al.
, 1990; Nossal, 1992; MacLennan, 1994; Kelsoe, 1996)
led us to initially explore the feasibility of modifying
immunization and fusion time lines used for developing
monoclonal antibodies. Our studies demonstrated
that monoclonal antibodies could be generated quickly
using an immunization and somatic fusion strategy,
which we refer to as repetitive immunizations, multiple
sites (RIMMS) (Kilpatrick et al.
, 1997). Immunizations,
somatic fusion, screening, and isolation of
affinity-matured IgG-secreting hybridoma cell lines
can be achieved within I month. RIMMS capitalizes on
somatic fusion of immune B cells undergoing germinal
center maturation in draining lymph nodes
(Kilpatrick et al.
, 1997, 2003). The immunization sites
used for RIMMS are proximal to easily accessible
regional lymph nodes. RIMMS involves the use of
P3X63/Ag8.653 murine myeloma cells stably transfected
with human BCL-2 (Kilpatrick et al.
Fusions can be performed as early as 7 days (Bynum et al.
, 1999) out to 14 days after the onset of immunization
using recombinant protein, conjugated synthetic
peptides, or drug haptens (Kilpatrick et al.
Ignar et al.
, 1998; Kinch et al.
, 1998; Wring et al.
Alligood et al.
, 2000; Ellis et al.
, 2000; Lindley et al.
2000). RIMMS has also been used successfully to
generate high-affinity antibodies using DNA-based
immunizations in conjunction with the PowderJect
gene gun (Kilpatrick et al.
, 1997, 1998, 2000, 2002, 2003;
Kinch et al.
This article describes the RIMMS procedure, including
the preparation of adjuvant, immunization,
isolation of lymph nodes, and our high-efficiency
polyethylene glycol (PEG)-induced somatic fusion
process. We also provide the reader with protocols for
developing a BCL-2-modified myeloma cell line.
II. MATERIALS AND
Fine curved forceps (Cat. No. 1-23-20) and microdissecting
scissors (Cat No. 11-250) are from Biomedical
Research Instruments. The following items are from
Corning Costar: 0.2-µm vacuum filter/storage units
(Cat. No. 431205), T 25-cm2
tissue culture flasks (Cat.
No. 3056), and 96-well tissue culture plates (Cat. No.
3595), as well as 24-well tissue culture plates (Cat. No.
3524). Items purchased from Sigma include Freund's
complete adjuvant (FCA) (Cat. No. F-5881), Hybri-
MAX azaserine hypoxanthine, 50× (Cat. No. A9666),
Hybri-MAX dimethyl sulphoxide (DMSO) (Cat. No.
D2650), deoxycholic acid sodium salt (Cat. No. D-
6750), and Igepal CA 630 (Cat. No. 1-3021, used in place
of NP-40). The following products are from InVitrogen:
0.45-µm nitrocellulose (Cat. No. LC2001), RPMI 640
(Cat. No. 11875-119), 10mM
, 100× nonessential amino
acids (Cat. No. 11140-050), L
with 10,000 units penicillin, 10,000 µg/ml streptomycin (Cat. No. 10378-016), gentamycin reagent, 50mg/ml
(Cat. No. 10131-035), 8-16% Tris-glycine gels (Cat.
No. EC6045), and SeeBlue Plus2 markers (Cat.
No. LC5925), as well as pcDNA3.1+ (Cat. No. V790-20),
Topo TA cloning (Cat. No. K4500-01), imMedia Amp
(Cat. No. Q600-20), imMedia Amp Agar (Cat. No.
Q601-20), and ethidium bromide (Cat. No. 15582-018).
NUNC 1.8-ml freezer vials (Cat. No. 66021-986),
as well as 10x phosphate-buffered saline (PBS) (Cat.
No. EX-6506), are from VWR. Defined fetal bovine
serum (FBS) (Cat. No. SH30070.03) is from Hyclone,
and Origen cloning factor (Cat. No. 210001) is from
Igen. JRH EX-Cell 610 HSF medium (Cat. No. 14610-
1000M) is from JRH Bioscience. The P3-X63Ag8.653
cells (ATCC CRL-1580) and PEG 1450, MW 1300-1600,
2-g bottle are from ATCC. The RIBI adjuvant (Cat. No.
R-700) is from Corixa. The anti-human BCL-2 monoclonal
antibody (Cat. No. M0887) is from Dako. The
following items are from Becton Dickinson: 1-ml Leur
Lok syringes (Cat. No. 309626), 5-ml Leur Lok syringes
(Cat. No. 309603), and 26G½ (Cat. No. 305111) and
16G1 (Cat. No. 305197) needles. BCIP/NBT color
development substrate (Cat. No. S3771) is from
A PTC-200 Peltier thermal cycler is from MJ
Research, Inc. (Cat. No. ALD-1244). DNA encoding
human bcl-2 is from Genecopoeia (Cat. No. GC-B0284).
Enzymes and lipid transfection reagents, Asp718 (Cat.
No. 814 245), Xba
I (Cat. No. 674 257), T4 DNA ligase
(Cat. No. 481 220), and FuGENE 6 (Cat. No. 1 815 091)
are from Roche. Software to analyze chromatographs
is from Gene Codes Corporation (Sequencer Cat. No.
SWC4.0). DNA isolation and cleanup kits are from
Qiagen, Inc. (QiaPrep spin DNA kit, Cat. No. 27106
and QiaQuick gel extraction kit, Cat. No. 28704).
Oligonucleotides are generated by Integrated DNA
Technologies, and Microspin S400 columns are from
Amersham Bioscience (Cat. No. 27-5140-01) for buffer
Eight- to 12-week-old female SJL mice are from
Jackson Laboratories, and BALB/c mice are from
Charles Rivers. Isoflurane (Iso Flo, Cat. No. 06-8550-
2/R1) is from Abbott Labs and is administered to mice
using a Vapomatic (Model 2) from AM Bickford, Inc.
A. Preparation of Culturing Media
B. Preparation of Antigen in Adjuvant
- Fusion selection medium, 1 liter: Combine 500ml
ExCell-610 HSF media, 260 ml RPMI 1640, 100 ml
Origen hybridoma cloning factor, 100 ml FBS, 10 ml L-glutamine/
pen-strep, 10 ml nonessential amino acids,
2 vials of azaserine hypoxanthine 50×, each reconstituted
with 10ml RPMI 1640. Sterile filter media in a
Corning 0.2-µm vacuum filter storage unit and store at
- Fusion selection medium without Origen cloning
factor, 1 liter: Follow the directions in solution 1, but
eliminate the Origen cloning factor and increase FBS
from 10 to 20%. This medium is used to eliminate background
from unfused B cells following somatic fusion.
- Fusion cloning medium, 1 liter: This medium is
used for limit dilution cloning of hybridomas.
Combine 500ml ExCell-610 HSF media, 280ml RPMI
1640, 100 ml Origen hybridoma cloning factor, 100 ml
FBS, 10 ml L-glutamine/pen-strep, and 10 ml nonessential
amino acids. Sterile filter medium in a
Corning 0.2-µm vacuum filter storage unit and store at
- Culturing medium for BCL-2-transfected P3-
X63Ag8.653 (ATCC CRL-1580) cells, 1 liter: Combine
890 ml RPMI 1640, 100 ml FBS, 10 ml L-glutamine / penstrep
solution, and 200µg/ml geneticin (G418). Sterile
filter medium in a Corning 0.2-µm vacuum filter
storage unit and store at 4 °C. One week before using
the cells for fusion, pass the cells into the justdescribed
medium without G418. The same medium
without G418 is used to culture the parental P3-
X63Ag8.653 myeloma cell line.
- Serum-free wash medium, 500ml: Add 5ml L-glutamine/
pen-strep solution to 495ml RPMI 1640,
sterile filter medium in a Corning 0.2-µm vacuum filter
storage unit, and store at 4 °C.
C. Immunization of Mice
- Dose per mouse: In a 1.5-ml Eppendorf tube, add
100µl of antigen diluted in sterile PBS to a final concentration
of 15µg for the primary immunization.
Add 100µl of RIBI adjuvant to the tube containing
the antigen and then vortex. Using a 1-ml Leur Lok
syringe outfitted with a 16G1 needle, remove 100µl of
Freund's complete adjuvant (vortex the vial of FCA
right before use).
- To make an emulsion for the primary immunization,
place the bevel of the needle against the inner
wall at the bottom of the Eppendorf tube containing
the antigen/RIBI mixture. Expel the Freund's complete
adjuvant from the syringe into the tube and then
draw the solution back up into the syringe holding the
bevel of the needle against the inner wall of the tube.
Repeat the process until a milky, slightly thickened emulsion is formed. Draw the emulsion back up into
the syringe. Carefully remove the 16G needle and
replace it with a 26 G ½ needle. The 300-µl volume is
then delivered to the six subcutaneous sites indicated
in Fig. 1, 50 µl per site, as detailed later.
- The final concentration of antigen used for the
secondary immunization is 5µg, and 2-5µg of the
antigen is used for the tertiary immunization. For secondary
and tertiary immunizations, dilute the antigen
in a 100-µl volume of sterile PBS, increase the RIBI
adjuvant volume to 200 µl, and eliminate FCA.
- For each antigen, immunize two 8- to 12-weekold
female SJL or two BALB/c mice (one mouse will
serve as a backup) at the sites indicated in Fig. 1 on
days 0, 7, and 10. Using this immunization time line,
fusion can be performed on day 11, 12, or 13. Alternatively,
mice can be immunized on days 0, 4, and 8, and
fusions can be performed on day 11. Fusions can also
be performed as early as day 7 by immunizing mice
on days 0, 2, and 4 (Bynum et al., 1999). The backup
mouse can be boosted every 2-3 weeks using conventional
protocols (see previous article).
- Anesthetize the mice with isoflourane for all
immunization time points.
- Inject 50µl of the antigen/adjuvant emulsion into
six sites proximal to axillary and brachial lymph nodes (thoracic region), superficial inguinal lymph nodes
(abdominal region), and popliteal lymph nodes
(located behind the knee) as indicated in Fig. 1.
|FIGURE 1 Immunization sites used for RIMMS are indicated by arrows. Following immunization, the
axillary, brachial, superficial inguinal, and popliteal lymph nodes are exposed and then removed aseptically.
Lymphocytes are isolated from the lymph nodes and are then used in a PEG-induced fusion.
D. Harvesting Lymph Nodes
E. Isolation of Lymphocytes from
- Perform euthanasia of mice using carbon
- Wet down the fur of the mouse with 70% ethanol.
- Lay the mouse on its back and lift up the skin in
the lower groin using 70% ethanol forceps. Using 70%
ethanol-rinsed microdissecting scissors, make a small
incision to open up the skin (do not cut open the
abdominal wall). To make a midsection incision, insert
the scissors under the skin and then make an incision
starting from the lower groin region up to the neck
using forceps to lift up the skin. Make incisions across
the top of the shoulders, down to the front feet on the
left and right sides. Then make incisions from the
lower groin area across to the top of the legs continuing
down to the hind feet. To expose the lymph nodes,
peel the skin back from the midsection incision, pull
back the skin from the hind legs, and secure with pins
as shown in Fig. 1.
- Using curved forceps that have been rinsed in
70% ethanol, remove the lymph nodes by placing
curved microforceps under each node and then pull up
gently to separate lymph nodes from surrounding
- Place the lymph nodes into a 60-mm sterile tissue
culture dish containing 5ml of serum-free wash
F. PEG-Induced Somatic Fusion
- In a laminar flow hood, remove the lymph nodes
from the 60mm tissue culture dish and transfer the
nodes into a new 60-mm dish containing 5ml of
serum-free wash medium. Fill a 5-ml syringe with
serum-free wash medium and add a 26G needle.
Gently hold individual lymph nodes with 70%
ethanol-rinsed curved microforceps. Insert the needle
into the node and then profuse with medium in order
to flush out the lymphocytes. Repeat process on each
node. Using two curved microforceps, gently tease
remaining cells from the capsules.
- Pipette the lymphocyte cell suspension into a
15-ml conical tube and allow the debris from the capsules
of the nodes to settle to the bottom of the tube
(less than 1 min). Pipette the cells away from the debris
and then transfer the cell suspension into a new 15-ml
conical tube. Count the cells (see article by Hoffman).
For each fusion, use 3 × 107 lymphocytes (see step 3 in
Note: The number of lymphocytes isolated from
pooled lymph nodes will range from 1-2 × 107 (weak
immunogen) to 1 × 108 when one female SJL mouse is
immunized using RIMMS. You can expect one-half of
this range when one female BALB/c mouse is used.
- Remaining immune lymphocyte cells not used
for fusion can be frozen back by pelleting the cells by
centrifugation at 400g for 5 min. Resuspend the cells in
1 ml of 90% FCS, 10% DMSO freezing media. Place
cells into 1 × 1.8-ml NUNC freezing vials labeled with
the antigen designation and the date. Place the vial
overnight at -80°C in a styrofoam container and then
transfer the cells to liquid nitrogen storage the following
day. These cells can be subsequently thawed and
used for somatic fusion.
G. Postfusion Care and Handling
- To affect a 1:1 lymphocyte-to-myeloma ratio,
harvest 3 × 107 P3-X63Ag8.653 (ATCC CRL-1580) or
P3X-BCL-2-transfected myeloma cells to be used for
the fusion in a 50-ml conical tube (see protocols on
how to generate a bcl-2-modified fusion partner in
Section IIIH). Check the viability of the myeloma cells
using trypan blue; ideally you want 90-98% viability Wash the cells in serum-free wash medium by centrifuging
at 400g for 5 min. Resuspend the cells in 2 ml
serum-free wash medium.
- To prepare the PEG for fusion (ATCC PEG 1450,
MW 1300-1600, 2-g bottle), very slowly heat the bottle
on a hot plate on a low setting, just enough to melt the
PEG. Do not boil the PEG. Using a 5-ml syringe outfitted
with a 16G1 needle, immediately add 3ml of
serum-free wash medium that has been prewarned to
37°C to make a 40% stock solution. Keep the PEG solution
in a 37°C incubator until you are ready to perform
the fusion. Use the PEG solution within 1.5h, as the
efficiency of the fusion will drop off significantly if you
use the PEG beyond 2h after preparation.
- In a 15-ml conical tube, mix 3 × 107 immune
lymphocyte cells isolated from lymph nodes with 3× 107 myeloma cells (from step 1). Centrifuge the cell
mixture at 400g for 5 min. Decant the media to leave a
"dry pellet" by removing as much media as possible.
Gently disrupt the pellet of mixed cells by tapping the
bottom of the tube.
- Using a 1-ml pipette, slowly add 300µl of the
PEG solution to the "dry pellet" of the myeloma and
lymph node cell mixture in the bottom of the 15-cc
tube. Mix gently and then allow the tube to incubate
in the hood for 5 min. Gently mix the cell suspension
again by tapping the tube and then allow the suspension
to incubate in the hood for another 5 min.
- Using a 5-ml pipette add 4 ml of the fusion selection
media to the fusion, resuspend the cells gently,
and then transfer the fusion suspension into a 250-ml
sterile bottle containing 196ml of fusion selection
media. Swirl gently to mix cell suspension. Plate out
the fusion in 10 × 96-well tissue culture plates by
adding 200-µl per well.
H. Generation of a P3X-BCL-2 Myeloma
Cloning of Human BCL-2
- Avoid removing the fusion plates from the incubator
for microscopic observation during the first few
days after the fusion.
- Within 7 days, remove one-half of the hybridoma
selection media and then replace with fresh hybridoma
selection media (containing azaserine hypoxanthine).
If you observe a high number of unfused lymphocytes
still growing, change the media to hybridoma selection
media containing 20% FBS, without Origen cloning
factor. Antibody from unfused B cells will give misleading
results in primary screening assays.
- Within 5-10 days you will be able to observe the
outgrowth of hybridomas in the fusion plates. Harvest
supernatant for ELISA analysis. ELISA positives are further tested in Western blot and immunoprecipitation.
- For limit dilution cloning for the isolation and
identification of monclonal antibody-producing cell
lines, please refer to the previous article.
- 10× TBE: 108g Tris base, 55g boric acid, 40ml
0.5 M EDTA (pH 8.0)
- Ethidium bromide: 10mg/ml in dH2O
I. Transfection, Isolation, and Identification of
BCL-2-Transfected Myeloma Cells
- PCR reaction mix: Mix 20pmol of primers (sense
5' cgg ggt acc gcc acc atg gcg cac gct ggg aga ac
3' and anti-sense 5' ccg tct aga tca ctt gtg gcc cag
ata ggc a 3'), human BCL-2 cDNA, 10× Advantage
PCR buffer, 200µM dNTP mix, dH2O, and
Advantage HF polymerase.
- Set parameters for human bcl-2 in a PTC-200
Peltier thermal cycler with the following conditions.
Cycle steps: a denaturation step of 3 min at
95°C to generate a hot start, 30 cycles at 95°C for
15s, 65°C for 15s, and then 72°C for l min, followed
by a soak step at 4°C.
- After amplification of the cDNA, separate the PCR
products and primers electrophoretically on a 1%
agarose/TBE ethidium gel and purify the 642-bp
band using the QiaQuick gel purification kit.
- Perform buffer exchange over a Sephadex-400
- Insert the cloned cDNA into pCR2.1 using the
TOPO TA cloning kit and transform into Top10 Escherichia coli.
- Prepare 6-10 cultures with 2 ml LB-amp media in
a 15-ml sterile culture tube. Pick a single isolated
colony with a sterile loop and inoculate each miniculture
with the isolated bacteria. Incubate and
shake overnight at 37°C.
- Isolate recombinants using the QiaPrep spin DNA
kit and digest 1 µg of the plasmid DNA with the
restriction endonucleases Asp718 and XbaI for 1 h
- Isolate the human BCL-2 cDNA fragment by excising
the 642-bp band and then subclone into a linear
pcDNA3.1(+) vector (digested with Asp718 and
XbaI) using T4 DNA ligase.
- Transform 2.5 µl of the ligation reaction into chemically
competent Top 10 E. coli and spread onto a
LB-amp agar plate. Incubate overnight at 37°C.
- Culture, isolate, and digest another 6-10 plasmid
DNA recombinants. Determine correct recombinants
by restriction endonuclease digests and
- Transfect pcDNA3.1(+) human BCL-2 plasmid
DNA into the P3-X63Ag8.653 cell line.
- RIPA buffer: 150 mM NaCl, 50 mM Tris, 1% Igepal,
0.25% deoxycholate, pH 7.5. To make 1 liter, add 8.76g
of sodium chloride, 6.35g Tris-HCl, 1.18g Tris base,
10ml Igepal, and 2.5 g deoxycholate to a total volume
of 1 liter. Sterile filter in a Corning 0.2-µm vacuum filter
storage unit and store at 4°C.
- Culturing medium for P3X63/Ag8.653 murine
myeloma cells: Combine 890ml RPMI 1640, 100ml FBS,
and 10 ml L-glutamine / pen-strep solution. Sterile filter
in a Corning 0.2-µm vacuum filter storage unit and
store media at 4°C.
- G418 selection medium, 1 liter, used for selection of
BCL-2- transfected P3-X63Ag8.653 cells (parental cells P3-
X63Ag8.653, ATCC CRL-1580): Combine 890ml RPMI
1640, 100 ml FBS, and 10 ml L-glutamine/pen-strep
solution. Add geneticin (G418) from 50-mg/ml stock
(potency is 600µg/mg) to final concentrations of
1 mg/ml, 500µg/ml, and 250µg/ml. Sterile filter in a
Corning 0.2-µm vacuum filter storage unit and store
media at 4°C.
- Culturing medium for BCL-2-transfected P3-
X63Ag8.653 cells, 1 liter: Combine 890ml RPMI 1640,
100 ml FBS, 10 ml L-glutamine / pen-strep solution, and
a final concentration of G418 at 200 µg/ml. Sterile filter
in a Corning 0.2%tm vacuum filter storage unit. One
week before using the cells for fusion, pass the cells
into the just-described media without G418.
- Phosphate-buffered saline: 10× PBS from VWR
(137mM NaCl, 2.7mM potassium chloride, 10mM phosphate buffer). To 100 ml of 10× PBS stock, add
900ml distilled water. Sterile filter in a Corning
0.2-µm vacuum filter storage unit.
- Hybridoma freezing medium: 90ml fetal bovine
serum, 10 ml Hybri-Max DMSO, sterile filter, and store
- Alkaline phosphatase developing buffer, pH 9.5 (0.1 M Tris HCL, 0.1M NaCl, 5 mM MgCl2): 1.52 g Tris-HCl,
10.94g Tris-OH, 5.85g NaCl, 1.15g MgCl2.6H2O dissolved
in 1 liter of distilled water, pH to 9.5, sterile
filter, and store at 4°C.
- 5% PBST Blotto: To 100ml of 1× PBST (0.05%
Tween-20), add 5 g powdered milk, mix well, and then
store at 4°C.
J. Western Blot Detection of Human BCL-2
- The day before transfection, plate P3X63/
Ag8.653 cells into 12 wells of a 24-well tissue culture
plate at a density of 4 × 105 cells per well in 2ml of
culturing media. Six of the wells will be used for transfection,
and 6 wells will be used for controls for the
G418 selection (see step 5). Incubate the cells overnight
in a 37°C, 5% CO2 humidified incubator. The cells
should be approximately 50% confluent.
- The DNA ratio used for transfection is 3:1. For
each well, use 0.5 ml of serum-free RPMI 1640 medium
containing 0.6µl of FuGENE 6 with 0.2µg of DNA
encoding human BCL-2.
- The following steps are taken to prepare the
FuGENE 6 reagent: DNA complex for transfection of
P3X63/Ag8.653 murine myeloma with the human
BCL-2 gene. Add 50µl of serum-free RPMI 1640 media
to a small sterile tube. Add 3 µl of FuGENE 6 transfection
reagent directly to the serum-free medium. Mix by
tapping the tube gently. Add 0.2µg of the DNA in a
volume of 1 µl to the tube. Mix the contents by tapping
the tube gently (do not vortex). Incubate the reaction
at room temperature for 15-30 min.
- Remove the medium from six wells of the plated
P3X63/Ag8.653 cells. Add 0.6ml culture medium to
each well. Using a sterile tip, add dropwise 53 µl of the
complex mixture from step 3. Gently mix the cell suspension
to disperse the reagent: DNA complex evenly.
Return the cells to the incubator.
- Within 24h, carefully remove medium from
wells that underwent transfection. To duplicate wells
add 2ml of the 1-mg/ml, 500-µg/ml, and 250-µg/ml
G418 selection media. To the remaining six wells that
were not transfected, add to duplicate wells 2 ml of the
1-mg/ml, 500-µg/ml, and 250-µg/ml G418 selection
media. These cells will serve as controls for the G418
selection process (cells should die).
- Within 4 days, remove selection media and
replace wells with fresh selection media. As the
selected cells grow, continue to maintain the cells
in selection media. Change the media every 3 to 5
- Expand the selected cell lines into T25-cm2 flasks
in the respective G418 selection media. Grow the cells
to 7-9 × 105/ml. Spin down the cells at 400g for 5 min.
Decant the supernatant, resuspend the cells in
hybridoma freezing medium, and aliquot 1 ml per 1.8-
ml Nunc freezing vials. Place the cells into a styrofoam
rack and then freeze overnight at -80°C. The next day,
transfer the cells to LN2. Freeze back stocks of the
transfected cell lines at 1 × 106 cells per vial in freezing
media. Continue to maintain the cells in culture in
respective selection media in order to prepare RIPA
extracts to determine the presence of the human BCL-
2 protein (see later).
- Following the transfections and selection of P3-
X63Ag8.653 cells with plasmid encoding human BCL-2,
maintain the myeloma cells in G418 selection media
containing final concentrations of 1 mg/ml, 500 µg/ml,
and 250µg/ml in T25-cm2 flasks. Also maintain the
parental P3-X63Ag8.653 cells, which will serve as negative
- Count the parental P3-X63Ag8.653 and the BCL-2
transfected cell lines using trypan blue (see article by
Hoffman). For each cell line, centrifuge 1 × 106 cells in
a 15-ml conical tube at 400g for 5 min.
- Remove all of the culture supernatant. To make
cell extracts in order to determine the presence of
human BCL-2 by Western blot, add 10µl of chilled
RIPA buffer to the cell pellet, resuspend the cells, and
then transfer the respective cell suspensions to 1.5-ml
Eppendorf tubes. Incubate tubes on ice for 15min.
- Microfuge the sample tubes in a microfuge set on
high for 15min at 4°C. Remove the supernatant
(discard the pellet) and then mix the supernatant 1:1
with 2× sample buffer. Heat samples at 96°C for 5 min.
- Load a 20-µl volume of the P3-X63Ag8.653 RIPA
extract (parental cell line) and each P3XBCL-2-
transfected cell extract onto individual lanes of a 1 × 10
well 8-16% Tris-glycine gel (InVitrogen) using one lane
for SeeBlue Plus2 markers. Run the gel for 90min at
125 V and then transfer the gel to nitrocellulose. Block
the nitrocelluose in 5% PBST Blotto overnight at 4°C.
- For detection of human BCL-2, add 10ml of antihuman
BCL-2 (Dako). Dilute 160 µl of the antibody per
10ml in 5% PBST Blotto blocking buffer. Incubate the
blot for 1 h at room temperature on a shaker or rocker
- Wash the blot four times for 5min with PBST.
Add 10ml of a 1:1000 dilution of goat anti-mouse IgGalkaline
phosphatase-labeled conjugate diluted in 5%
PBST Blotto blocking buffer to the blot. Incubate for
1 h on a rocker platform.
- Wash the blot four times with PBST. Develop the
blot using 10ml alkaline phosphatase buffer containing
66 µl of NBT and 33 µl of BCIP. Immerse the blot
into the developing substrate, place on a rocking platform,
and incubate until a purple color develops. Stop
the reaction by removing the developing substrate and
rinsing the blot with ddH2O.
- Figure 2 demonstrates a Western blot indicating
the presence of BCL-2 in RIPA extracts made from transfected, G418 selected P3-X63Ag8.653 myeloma
- Expand the uncloned cells from wells that
demonstrate the presence of BCL-2 into a 75-cm2 tissue
culture flask containing 35 ml of media supplemented
with 250µg/ml G418. Grow the cells to 7-9 × 105/ml.
Spin down the cells at 400g for 5min. Decant the
supernatant, resuspend the cells in hybridoma freezing
media, and aliquot 1 ml per 1.8-ml Nunc freezing
vials. Place the cells into a styrofoam rack and then
freeze overnight at -80°C. The next day, transfer the
cells to LN2.
|FIGURE 2 Titration of RIPA extracts of P3-X63Ag8.653 clones
(ERIC5, ERIC9, and ERIC10) stably expressing human BCL-2, as well
as uncloned parental BCL-2 transfected cells, indicates the presence
of human BCL-2 by Western blot using an anti-human bcl-2-specific
monoclonal antibody. Control untransfected P3-X63Ag8.653 cells
serve as a negative control.
K. Limit Dilution Cloning of P3Xbcl-2
- Count the P3XBCL-2 cells (see article by
Hoffman) and then dilute the cells to 330 cells in a total
of 200ml of culturing media containing 250µg/ml
G418. To increase the limit dilution cloning efficiency,
the Origen cloning factor can be added to the G418
- Add 200µl of the cell suspension per well to 10
× 96-well sterile tissue culture plates.
Incubate the cells at 37°C in a humidified incubator.
- Microscopically scan the wells to identify one cell
per well within 48h of plating. Mark the wells that
have clones derived from single cells.
- Once the clones have grown to cover 50-75%
of the well, expand the cells into 24-well plates containing
2ml per well culturing media containing
200µg/ml G418. Allow the cells to grow to 80-90%
confluency in 6 wells. Remove cells from 3 wells and
make RIPA lysates using the aforementioned procedure.
Test for expression of BCL-2 using the Western
blotting procedure. (Fig. 2).
- Expand the cells that exhibit high BCL-2 expression
levels to T25-cm2 tissue culture flasks. Freeze back
master stocks as detailed earlier.
- Test the P3XBCL-2-transfected cells for their
ability to negatively select (die) by placing the cells in
fusion selection media containing HybriMax azaserine
hypoxanthine. Due to the presence of BCL-2, the cells
will take approximately 2-3 days longer to die as compared
to the parental untransfected P3-X63Ag8.653 cell
Unfused B cells producing antigen-specific immunoglobulin
can contribute to background, which may
indicate false positives in a primary ELISA screen. It
is imperative to change the media on fusion plates one
to two times before screening (see Section IIIG, step
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