Table Of Content1
Isolation and Expansion of HIV from Cells
and Body Fluids by Coculture
James R. Lane
1. Introduction
HIV can be recovered from infected patients at all stages of the disease spec-
trum. Typically, the quantity of biologically active virus, or viral protein, in
body tissues is below the level of direct detection by either antigen capture or
reverse transcrtptase assays.C onsequently, the virus must be expanded in cul-
ture. This may be achieved by the coculttvation of pattent material with mito-
gen-stimulated peripheral blood mononuclear cells (PBMCs) from normal,
healthy donors. These cocultures are then maintained by regularly scheduled
mterleukin-2 (IL-2) supplemented medium replacement, and the periodic
addition of freshly stimulated normal donor PBMCs. Durmg this cocultivation
period, culture fluids are harvested at regular intervals and tested for the pres-
ence and subsequent replication of HIV. Cultures fatling to demonstrate evi-
dence of vnus expression within 35 d are usually terminated.
It is well recognized that the efficiency of virus isolation is dependent on
specimen type. Patient PBMCs represent a useful source of patient vnus, how-
ever, isolates can be obtained from such body fluids as spinal fluid or milk. In
addition, disease state also influences the isolation of vnus. Isolation rates
reported for patients with AIDS are consistently higher than from pre-AIDS
patients Using the Walter Reed classification system, Burke et al. (1) demon-
strated vn-us isolation rate increases with CD4 cell depletion and impaired
delayed hypersensitivity. Furthermore, these authors also noted that the time
required for virus isolation decreased with disease progression.
To mmimize genetic drift (selection) during the periods of virus isolation, it
is crttical that these procedures be completed as quickly as possible. This pro-
From Methods m Molecular Medrcme, Vol 17’ HIV Protocols
Edlted by N L Mtchael and J H Kim 0 Humana Press Inc , Totowa, NJ
3
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cess is facilitated by utilizing target PBMCs from donors prescreened for eff-
cient virus isolation and expansion. In the author’s laboratory, PBMCs from
these selected donors are routinely cryopreserved and reserved specifically for
studies. This practice of standardizing assayc oculture conditions not only muu-
mizes experimental variation but also limits the opportutnty for genetic drift of
viruses during prolonged expansion periods and the use of PBMCs from mul-
tiple donors. The establishment and culturing procedure for isolating and ex-
panding HIV-l from a variety of patient specimen types is described below.
Although vu-us isolation and expansion can also be performed using contmu-
ous cell lmes, the focus of this chapter will be with the use of PBMCs.
2. Materials
2.1. Donor PBMC
Normal donor buffy coats (leukocyte-enriched whole blood, from an HIV
negative donor), also known as leukopacks, may be obtained from a blood
bank or donor service for processing within 12 h of collection. The donors
should be screened by the standard assays currently required by the Food and
Drug Administration (FDA) and the American Association of Blood Banks
(AABB). To obtain the PBMCs from the leukopacks, follow the same proce-
dure described for patient whole blood samples (Subheading 3.1.)
2.2. Continuous Cell Lines
There are numerous cell lines available through the AIDS Research and
Reference Reagent Program (Division of AIDS, NIAID, NIH) suitable for HIV-
infection and expansion of virus. In this work, H9 cells are used as an example.
2.2. Reagents
1 RPM1 1640 (Gibco [Gaithersburg, MD] or equivalent)
2. Fetal bovine serum (FBS) (Gibco or equivalent) (heat Inactivated, 56°C 30 mm)
3. Pen-Strep (Gibco or equivalent) Concentratton = 10,000 U Pen and 10 mg strep/ml
4. Ficoll-Paque (Pharmacia or equivalent).
5. Trypan blue: 0 4% (Gibco or equivalent).
6. PHA (Murex cat #HA 17 or equtvalent, 10 mg lyophyhzed powder)
7. L-Glutamme (200 mM) (Grbco or equivalent)
8 IL-2 (Boehrmger-Mannheim [Indianapolis, IN] cat# 663-58 1, or equivalent). 200 U/mL.
9 Polybrene (Sigma [St Louis, MO] or equivalent).
10. DMSO (Sigma or equivalent)
2.3. Reagent Preparation
1. Ftcoll wash medium: RPM1 1640,2% FBS, 1% Pen-strep. Filter stertltze using a
0.2-u filtration unit. Store at 4°C + 2’C Discard after 2 wk.
Isolation and Expansion of HIV from Coculture 5
2 Complete medium: RPM1 1640, 15% FBS, 10% IL-2,1% Pen-strep, 2 rnA4
L-Glutamine Filter sterilize using 0 2-punit. Store at 4°C + 2°C. Discard after 2 wk
3. PHA stock solution: Reconstitute PHA (10 mg bottle) with 10 mL RPM1 1640.
Filter stenhze using syringe filter. Final concentration is 1000 pg/mL. Ahquot
0 34.5 mL into cryovlals Store at -20°C + S’C. Discard after 1 yr.
4. Polybrene stock solution (0.5 mg/mL): Dissolve 100 mg of polybrene into 200 mL
RPM1 1640, filter sterilize using a 0.2-p filtration unit. Store at 4°C f 2°C
Aliquots are stable for 1 yr.
5. Coculture medium: RPMI-1640, 15% FBS, 10% IL-2, 1% Pen-strep, 2 mM
L-Glutamme, 2 clg/mL polybrene. Store at 4°C + 2°C. Discard after 2 wk.
6. H9 medium. RPMI-1640, 15% FBS, 1% Pen-strep, 2 mM L-Glutamme, 2 &mL
polybrene. Store at 4°C * 2°C. Discard after 2 wk.
3. Methods
3.7. Isolation of PBMC from Whole Blood
1 In a biologlcal safety cabinet (BSC), dilute one part of fresh whole blood sample
with two parts Ficoll wash medium in a 50-mL centrifuge tube (CT) (see Notes 1
and 2). For example, 10 mL of whole blood would be added to 20 mL of wash
media. Mix gently by pipeting slowly up and down three to four ttmes.
2. Transfer no more than 30 mL of diluted blood into a 50-mL CT. Slowly layer 10
mL of Ficoll solution underneath the diluted blood mixture by placing the tip of a
lo-mL pipet containing the Flcoll at the bottom of the CT (see Note 3)
3 Securely replace cap on the 50-ml CT. Centrifuge for 30-35 min at 40&44Og
with no brake at room temperature.
4. In a BSC, using a 5- or IO-mL plpet, remove the top layer (I.e., diluted plasma
and most platelets) to just above the “buffy coat” and discard. Using a different
5-mL pipet each time, collect the “huffy coat” cells from each gradient tube and
transfer to a 50-mL CT. “Buffy coats” from two gradients may be pooled into one
50-mL CT provided they are from the same patient or leukopack.
5. Add wash medium equaling 2-3 times the vol of the “buffy coat” layer Centri-
fuge for 15-20 mm at 220-240g at room temperature
6. Decant supernatant, and resuspend cell pellet with 20 mL of wash medium
Remove the appropriate amount of cell suspension to perform a cell count using
either a hemocytometer or an electronic countmg device.
7. Take 0.1 mL of the cell suspension, and mix with 0.1 mL of 0.4% trypan blue (see
Note 4). Allow dilution to incubate 3-5 min at room temperature (see Note 5).
8 Apply 0.025 mL of the trypan blue/cell mixture to a hemacytometer This vol-
ume ~111 ensure that the hemocytometer is not overfilled Remove hemocytom-
eter from the BSC and place it on the stage of a binocular microscope and focus
on the cells.
9. Count the unstained (viable) and stained (nonviable) cells separately m the
hemocytometer. Count to a total of 100 cells.
10. Calculate the percentage of viable cells as follows:
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viable cells (%)= total number of viable cells counted x 100
total number of cells counted
11. Based on the cell count/ml obtained in step 6, multiply the number by the %
viability. For example, tf a cell count of 3 x 106/mL was obtained and the vtabil-
tty is 73.3%, the actual cells available equals 2.2 x lo6 (3 x 0.733 = 2.2)
12. Determine the amount of cells needed for the protocol of interest For example, m
the vnus isolation protocol (Subheading 3.3), 3.3 x lo6 cells are required Divide
cells needed m the parttcular protocol by the number of viable cells/ml to obtam
the amount m mL of cell suspension required. In the example used here, this would
be 3.3 x lo6 cells required divided by 2.2 x lo6 viable cells/ml As a result, 1.5 mL
of the cell suspension prepared above is needed m this particular example
13. Remove volume (m the example cited tt would be 1.5 mL) and place m a 15-mL
conical centrifuge tube.
14 Centrifuge for 10-15 mm, at room temperature, 22&24Og Decant supernatant
The cell pellet is now ready for use m the protocol of interest
3.2. PHA Stimulation of Normal Donor PBMC
1, Obtain enough donor cells (prepared as described m Subheading3.1.) for the appropn-
ate protocol As an example, assume that 60 x lo6 cells will be PHA strmulated.
2 Transfer the cells into a single 15-mL CT containing 10 mL of wash media
3. Centrifuge at 22&24Og for 1 O-l 5 mm at room temperature Decant the superna-
tant and discard.
4 Gently resuspend cell pellet m 8.0 mL complete medium, by slowly drawing
cells up and down m a 10 mL ptpet, transfer the cell suspension into a T-75 flask.
5 The T-75 flask now contams approx 60 x lo6 cells m 8.0 mL complete medium
Add 40 & of stock PHA to each flask.
6. Incubate at 37’C f 2°C for 15-30 min. Stand flask upright. After mcubatton, add
12 mL complete medium to each flask. Incubate in upright position for 18-24 h
m a CO2 incubator (5% CO* and 95% humidity) at 37°C f 2°C
7 After 18-24 h incubation, proceed as follows:
a. Transfer the cell suspension from each flask mto a 50-mL conical CT Centri-
fuge at 220-240g for 1 O-l 5 min at room temperature.
b Decant supernatant and discard. Resuspend each cell pellet m 10 mL of com-
plete medium and divide equally mto two new T-75 flasks (1:2 split) or one
new and reuse old/origmal flask
c. Add 25 mL of fresh complete medium to each flask Incubate m an upright
position m a CO* incubator (5% CO, and 95% humidity) at 37’C + 2’C for an
additional 48-72 h.
8. Cells are now ready for use m the appropriate protocol
3.3 Virus isolation from PBMC by Tube Coculture
1 A total of 3.3 x lo6 patient PBMCs in 30 pL of coculture medmm is needed for
this assay. On setup day for cocultures, a total of 4 x lo6 PHA-stimulated PBMCs
Isolation and Expansion of HIV from Goculture 7
is needed for each patient sample tested. At d 314 and at d 7 intervals ( i.e., d 7,
14,2 1, and 28), an additional 4 x lo6 PHA-stimulated PBMCs will be needed.
2 Gently resuspend the cell suspension containing 3 3 x lo6 patient PBMCs with
an additional 300 pL of coculture medium by pipeting slowly up and down 3-4
times using a digital plpetor and 200-1000~pL pipet tip. Total volume should
now equal approx 330 pL. Add 300 JJL of this patient PBMC suspension to one
coculture tube labeled “A 1”. Add the remaining 30 pL of patient PBMC suspen-
sion to one coculture tube labeled “A2”. To this latter tube (“A,“), add 270 p.L of
coculture medium
3. Add 2 x lo6 PHA-stimulated PBMCs in 200 pL of coculture medium to each
coculture tube.
4 Incubate cocultures at 37OC f 2’C!, 5% CO,, and 95% humidity for 30-40 mm.
5. Add 2.0 mL coculture medium to each coculture tube Gently mix cell suspen-
sion by plpetmg up and down slowly 2-3 times.
6. Incubate at 37°C _+2 ”C, 5% COz, and 95% humldlty.
7 On d 3 or 4, gently remove 2.0 mL of culture supernatant without disrupting the
cell pellet and discard Add 2.0 mL of coculture medium containmg 2 x lo6 PHA-
stimulated PBMCs to each culture tube
8. On d 7, 14,21,28, and 35, remove 2.0 mL of culture supematant without disrupt-
ing the cell pellet. Reserve 700-1000 pL of supematant for HIV p24 assay Dls-
card remaining supematant (see Note 6).
9. On d 7, 14, 21, 28, and 35, add 2 x lo6 PHA-stimulated PBMCs m 2.0 mL of
coculture media
10 On d 10, 17,24, and 3 I, remove 2 0 mL of culture supematant wlthout disrupting
the cell pellet and discard Add back 2.0 mL of coculture medium
11 Terminate negative cocultures after obtaining p24 results of d 35 culture
supematants
3.4. Patient Spinal Fluid (CSF) Isolation Procedures
1. Centrifuge tube containing CSF at 9OOg for 15 mm.
2. Carefully remove the CSF supematant from the tube and transfer to another tube
Do not disturb the cell pellet
3. Resuspend the cell pellet using 200 pL of CSF supematant removed in step 2
Transfer resuspension to a culture tube. Add 0.2 mL PHA stimulated normal
donor PBMCs (2 x 106) and mix gently Incubate at 37’C for 15 mm, then add
coculture medium for a final vol of 2.5 mL. Incubate at 37”C, feed, and sample as
described in Subheading 3.3.
4 Save tissue culture fluids from cocultures collected at weekly intervals (d 7, 14,
etc.) for subsequent p24 antigen capture ELISA. Maintain culture vol at 2.5 mL
3.5 Patient Milk Isolation Procedures
The procedure for processmg milk samples and establishing cocultures for
virus (HIV) isolation mvolves using both the cellular andl iquid portions of the
milk. To increase success m Isolating virus a total of six tube cultures are set up
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for each specimen: A 1, A2, and A3 from milk suspension and Cl, C2, and C3
from milk cells.
1. Culture of milk suspenston
a. Mix milk suspenston gently, remove 430 pL milk to set up Al, A2, and A3
tube cultures. Save the remammg suspenston for Part B. Set up cultures as
follows: Transfer 300 $ milk into a culture tube labeled Al, Then transfer
100 & mtlk mto a culture tube (A2) + 200 I.~L coculture medium. Finally,
transfer 30 pL milk mto a culture tube (A3) + 270 pL coculture medium
b. To each culture tube, add 2 x lo6 (200 pL) PHA-stimulated normal PBMCs,
Incubate at 37°C for 30 min.
c. After incubation, add 2.0 mL coculture medium to each tube culture and con-
tinue mcubation. Feed (replace medium) and add fresh PBMCs cells as
described (see Subheading 3.3.).
e. Save tissue coculture flmds collected weekly (day 7, 14, etc ) for subsequent
p24 antigen capture ELISA. Maintain culture vol at 2.5 mL.
2. Culture of milk cells:
a Centrifuge remaining milk at 400g for 15 min. Label three culture tubes C 1,
C2, and C3.
b. Carefully remove mtlk supernatant without dtsturbmg cell pellet
c. Resuspend cell pellet in 333 & of coculture medmm; then set-up cultures as
follows: Cl contains a 300 pL cell suspension, C2 contains a 30 pL cell sus-
pension + 270 & coculture medium, and C3 contains 3 r.lc cell suspension +
295 pL coculture medium
d To each culture, add 2 x lo6 (in 200 pL) PHA-stimulated normal PBMCs, and
Incubate 37°C for 30 mm
e. After mcubatton, add coculture medium for a final vol of 2 5 mL m each
culture tube, and continue incubation. Feed (replace medmm) and add PBMCs
as described (see Subheading 3.3).
f Save tissue coculture fluids collected weekly (day 7, 14, etc ) for subsequent
p24 antigen capture ELISA. Maintain culture vol at 2.5 mL.
3.6. Expansion of HIV Isolated by Tube Culture
1 Begin expansion when tube cultures demonstrate posmvity
2 Gently mix by pipetmg up and down 34 times Remove 1.25 mL, and place mto
a new coculture tube labeled “al” or “a2” as appropriate to indicate expansion
from the original Al or A2 tube.
3. Add 2 x lo6 PHA-sttmuiated PBMCs m 0.2 mL of coculture medmm to each of
the tubes. Then add 1.25 mL of coculture medium to each of tubes
4. Incubate at 37°C & 2”C, 5% COz, and 95% humidity.
5 Four days after mmatmg expansion, remove 2 0 mL of culture supematant from
the tubes wtthout dtsruptmg the cell pellet. Reserve some of the supematant for
p24 testing. Add 2.0 mL of the coculture media back mto each tube, Incubate at
37°C + 2”C, 5% C02, and 95% humldtty.
Isolation and Expansion of HIV from Coculture 9
6 Seven days after initiating expansion, remove 2.0 mL of culture supernatant from
the tubes wlthout dlsruptmg the cell pellet Reserve some of the supernatant for
p24 testing (see Note 6). Add 2 x lo6 PHA-stimulated PBMCs in 0 2 mL of
coculture medium. Then add 2 0 mL of coculture medium. Incubate at 37’C f
2”C, 5% CO,, and 95% humldlty.
7 Continue replacing media and adding cells every 34 d until positive cultures
have been reestablished When the culture has been determined to be positive,
remove 2.0 mL of supernatant Add 2 x lo6 PHA stimulated PBMCs m 0.2 mL of
coculture medium Then add 2.0 mL of coculture medium. Incubate at 37’C +
2”C, 5% CO,, and 95% humidity for 34 more days.
8 Resuspend cell culture, and pool all tubes from a particular patlent mto a 15-mL
centrifuge tube.
9 Centrifuge at 300-340g for 15-20 min at 4°C f 2°C.
10. Remove supernatant and place into cryovlals for storage
11. If desired, resuspend cell pellet m complete media with 10% DMSO and allquot
mto cryovials for storage
12 Place all cryovlals mto a hquld nitrogen storage tank
3.7. HIV lnfecfion of Continuous Cells
1 H9 cells are cultured m H9 medmm and incubated at 37°C f2”C, 5% CO,, and
95% humidity until ready for use (see Note 7)
2 Obtain 3 x lo6 H9 cells and prepare as a cell pellet m a 15-mL centrifuge tube
Carefully remove the H9 medium without disturbing the cell pellet.
3 Resuspend the cells in 0 5-l mL of virus supernatant and incubate at 37“C for
30 mm Durmg this mcubatlon, gently shake the centrifuge tube every 10 min to
resuspend the cells
4 Centrifuge cell suspension at 220-240g for 10-15 min at room temperature.
Remove the supernatant and discard.
5 Add 2 5 mL H9 medium to the tube. Gently mix cell suspension by plpetmg up
and down slowly 2-3 times. Transfer cell suspension to a coculture tube
6. Incubate at 37°C f 2”C, 5% CO,, and 95% humidity.
7. On d 3 or 4, gently remove 2.0 mL of culture supematant and reserve 700-1000 pL
for HIV p24 assay. Add 2.0 mL of H9 medium containing 2 x lo6 H9 cells
8. Repeat step 7 until positive culture obtained (see Note 8)
Notes
1. All solutions and equipment coming mto contact with cells must be sterile, and
proper sterile technique must be used accordmgly. All medium and Ficoll-Paque
are to be used at room temperature. When handling patient samples or viral cul-
tures, all work must be performed m a BSC.
2. Specimen required IS a whole blood sample m anticoagulant. A minimum amount
of blood that will yield 3.3 x lo6 PBMCs is required. For most subjects, 7-10 mL
of blood 1s sufficient The blood should be processed in the laboratory within 24 h
of draw. Samples showmg hemolysis are not acceptable.
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3. To maintain the Ficoll-blood interface, it is helpful to hold the CT at a 45” angle.
4 Trypan blue is one of several stains recommended for use m dye exclusion proce-
dures for viable cell counting. This method is based on the prmciple that hve
(viable) cells do not take up certain dyes, whereas dead (nonviable) cells do.
5 Cells should be counted within 3-l 5 mm of mixing with trypan blue, since longer
incubation periods will lead to cell death and reduced vtabtlity counts.
6 A successful virus isolation has occurred when two consecutive cultures are posi-
tive for the presence of p24
7. For successful mfection, H9 cells should be m logartthmic growth and be highly
viable
8. If large volumes of virus are desired, transfer the cell suspension to a T-25 flask
and maintain the culture at a H9 cell concentration of 2 x lo6 cells/ml
References
1. Burke, D. S, Fowler, A K , Redfield, R R , Dilworth, S., and Oster, C.N (1990)
Isolation of HIV-l from the blood of seroposmve adults: patient stage of illness
and sample inoculum size are major determinants of a positive culture J AIDS
3(12), 1159-l 167
Quantitative HIV Culture
James R. Lane
1. Introduction
This procedure is used for establishing cocultures for virus Isolation by an
endpoint dilution assay m a 96-well tissue culture plate. This is a cell culture
assay based on the detection of HIV in patient peripheral blood mononuclear
cells (PBMC) by the appearance of p24 gag protein m the culture supernatant.
It can be used m a research setting as a measure of viral burden because the
number of patient cells required to produce a posittve culture IS indicative of
cellular viral load.
2. Materials
2.1. Donor PBMC
Normal donor buffy coats (leukocyte-enriched whole blood, from an anti-
HIV negative donor), also known as leukopacks, may be obtained from a blood
bank or donor service for processing within 12 h of collection. The donors
should be screened by the standard assays currently required by the Food and
Drug Administration (FDA) and the American Association of Blood Banks
(AABB). To obtain the PBMC from the leukopacks, follow the same proce-
dure described in Chapter 1 [Isolation and Expansion of HIV from Cells and
Body Fluids by Co-Culture], Section 3.1.
2.2. Reagents
1. RPM1 1640 (Gtbco [Gaithersburg, MD] or equivalent).
2. Fetal bovine serum (FBS) (Gibco or equivalent) (heat-inactivated, 56”C, 30 mm).
3. Pen-Strep (Gibco or equivalent) Concentration = 10,000 U Pen and 10 mg strep/ml
4. Ficoll-Paque (Pharmacla or equivalent).
5. 0.4% Trypan blue (Gibco or equivalent).
From Methods fn Molecular Medmne, Vol 17 HIV Profocols
EdIted by N L Michael and J H Kim 0 Humana Press Inc , Totowa, NJ
11
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6. PHA (Murex cat #HA I7 or equivalent, IO mg lyophylized powder)
7 200 ti L-Glutamme (Gibco or equivalent)
8 200 U/n& Interleukin-2 (IL-2) (Boehringer-Mannheim cat# 663-58 1, or equivalent)
9. Polybrene (Srgma [St Louis, MO] or equivalent)
2.3. Reagent Preparation
1 Fmoll wash medium* RPM1 1640,2% FBS, 1% Pen-Strep Filter sterilize using a
0 2 u filtration unit Store at 4°C f 2°C Discard after 2 wk
2 Complete medmm* RPM1 1640, 15% FBS, 10% IL-2,1% Pen-Strep, 2 mM L-
Glutamme Filter sterilize using 0 2 u filtration unit. Store at 4°C + 2°C. Discard
after 2 wk
3 PHA stock solution Reconstitute PHA (10 mg bottle) with 10 mL RPM1 1640
Filter sterilize using syringe filter Final concentration is 1000 pg/mL Altquot
0 3-O 5 mL mto cryovrals. Store at -20°C _+5 °C Discard after 1 yr.
4 Polybrene stock solution (0 5 mg/mL) Dissolve 100 mg of polybrene into 200 mL
RPM1 1640, filter sterilize using a 0 2 u filtration umt Store at 4’C + 2°C
Ahquots are stable for 1 yr.
5. Coculture medium* RPMI-1640, 15% FBS, 10% IL-2, 1% Pen-strep, 2 mM L-
Glutamme, 2 ug/mL polybrene Store at 4°C f 2°C Discard after 2 wk
3. Methods
1 Label four sterile cluster “A”, “B”, “C”, and “D”
2 Resuspend a cell pellet contammg 2 2 x lo6 patient cells processed as described
in Chapter 1 [Isolation and Expansion of HIV from Cells and Body Fluids by
Coculture], Section 3 1 , m 350 pL of coculture medium Gently mix suspension
2-3 times using a 200-l 000-pL pipet tip.
3 Transfer all of the resuspended cell pellet from step 2 mto Tube A
4. Gently mix and transfer 120 pL of cell suspension from Tube A mto tube B
containing 260 pL of coculture medium.
5 Gently mix and transfer 120 $ of cell suspension from Tube B mto Tube C
containing 260 pL of coculture medium
6 Gently mix and transfer 120 pL of cell suspension from Tube C mto Tube D
contammg 260 pL of coculture medium
7 Dispense four replicates of each cell dilution mto a 96-well mrcrotrter plate ac-
cording to the format m Table 1.
a. 50 pL of Tube A cell suspension mto wells 3C-3F
b. 50 pL of Tube B cell suspenston mto wells 4CXF.
c. 50 pL of Tube C cell suspension mto wells 5C-5F.
d. 50 pL of Tube D cell suspension to wells 6C6F.
8. Obtain 4 0 x lo6 PHA stimulated, viable, PBMC prepared as described m Chap-
ter 1 [Isolation and Expanston of HIV from Cells and Body Fluids by Coculture],
Section 3 )
9. Centrifuge at 22&24Og for 10-15 mm at room temperature. Decant supernatant
10 Resuspend pellet in 2 0 mL of coculture medium
