Box 1|
Videomicroscopy and functional analysis of haematons in long-term organotypic culture
(Hem-LOC)
(i) Short-term and long-term myeloid cultures
1) Plate ~50-100 haematon units in 0.8 ml My-LTCM into a hydron-bordered, 25-sq cm flask for 48 h
to force initial adherence in the CO2 incubator at 37 °C.
2) Remove spent medium carefully, add 5 ml fresh medium and return the culture in the incubator
for 2-4 h.
3) Close the flask hermetically and install under the inverted microscope at 33 °C.
4) Select representative haematons and film over time through a digital camera.
▲CRITICAL STEP The choice of frame characteristics (region of interest, magnification, acceleration of
motion and length of video) should be adapted to the thickness and mobility of haematopoietic cells
underneath the stromal cells.
5) Analyse the cellular and molecular composition of adherent haematon islands at increasing time
as shown in Box 2|.
(ii) Myelo-lymphopoietic switch culture37
6) Maintain haematons in My-LTCM for 2 -to -5 w at 33 °C.
▲CRITICAL STEP 2-5 µM hydrocortisone ablates lymphopoiesis and allows myelopoiesis from
pluripotent HSCs.
7) Rinse the adherent layer gently 3 -times and then switch the culture-conditions to B-cell growth.
▲CRITICAL STEP The use of RPMI-1640 with 10 mM HEPES, 5x10-5 M βMeEtOH and preselected or
commercially certified 10% (vol/vol) FCS is recommended.
8) Continue the cultures for an additional 3-4 w.
9) Identify B-lymphocytes emerging in the cobblestone islands from 1 w after the myelolymphopoietic switch by virtue of their focal proliferation both underneath and on the top of stroma
cells.
10) Phenotype B-lymphocytes in the non-adherent compartment by FACS and in newly formed B-cell
foci by in situ immunocytochemistry using the CD19/CD20 markers.
?TROUBLESHOOTING The CD19 antigen is lost upon collagenase digestion.
▲CRITICAL STEP Analyze CD19+ cells in the adherent layer by FACS using mechanic removal and nonenzymatic dispersion.
Box 2|
Blotting haematons in alginate for polychromatic staining and 3D LSCM imaging
(i) Blotting native haematon isolates
▲CRITICAL STEP
1) Fix haematons in Zn-7 for 4-6 h at 4 °C. Use 4 ml fixative for one femur equivalent haematons.
2) Remove the fixative then add 4 ml Ca2+-free 100 mM Tris-HCl (pH7.4) to the pellet. Leave particles
to settle for 10 min and then repeat the washing twice.
3) Stirr the haematon pellet in 1 ml Tris-HCl buffer and then mix with 1 ml 4% (wt/vol) alginate.
4) Transfer the pre-polymer on a cell strainer (BD Falcon, 40 µm mesh, 20 mm diameter nylon filter)
to collect haematons in a fine sheet of viscouse alginate.
▲CRITICAL STEP Verify the even repartition of haematons on the filter and displace them if required
under the stereomicroscope.
5) Place the filter on top of 4 ml 10 mM CaCl2 in 100 mM Tris-HCl (pH7.4) to polymerize the hydrogel.
●TIMING 30 min.
6) Turn the filter upside down and remove the excess hydrogel from the backside using a spatula.
7) Return the filter and wash the jellified thin haematon layer in 100 mM Tris-HCl (pH7.4) containing
10 mM CaCl2. Cut the filter into several pieces using fine scissors under the stereomicroscope and
transfer these fine sheets into separate wells for staining.
The alginate gel depolymerises in the absence of Ca2+. In order to conserve the integrity of
alginate sheets add 10 mM CaCl2 to all working solutions.
8) Wash haematons three times (20 min each) in 100 mM Tris-HCl with 10 mM CaCl2.
9) Proceed with the required histochemical or immunofluorescence staining protocol: for
simultaneous staining transfer smaller samples in 24-well plates and use 150 µl reaction volumes.
Larger sheets can be transferred and processed in 12-well plate using 400 µl volume.
●TIMING Usually 2-4 d depending on the number of staining reactions.
(ii) Blotting plastic adherent haematons from organotypic culture
▲CRITICAL STEP Haematons adhere to plastic and form flattened islands within two to three days in
culture. The whole adherent layer can be recovered and processed for multicolour staining using
alginate, due to the good penetration of the pre-gel followed by slight shrinkage during
polymerization. Reagents are calculated for a 25-sq cm culture dish.
1) Remove the culture medium, add 5 ml Zn-7 fixative for 5 min then replace the first, -spent- fixative
with 5 ml new fixative for 4-6 h at 4 °C.
2) Wash the adherent layer gently, three times for 15 min in 100 mM Tris-HCl (pH7.4).
3) Optional: For detecting intracellular proteins and extracellular matrix components one may apply
at this point the second fixative (acetone:methanol/1:1 vol/vol) for 10 min at -20 °C. Wash the
adherent layer twice in 100 mM Tris-HCl.
4) Remove the washing buffer then add 1.5 ml 2% (wt/vol) alginate in 100 mM Tris-HCl (pH7.4) buffer
per 25-sq cm culture dish. Spread the pre-gel evenly over the adherent layer then let the dish
horizontally for 4-16 h at 4 °C.
5) Add carefully 3 ml 50 mM CaCl2 in 100 mM Tris-HCl to induce gelling. Place the dish at 4 °C for 1 h.
At this point the alginate retracts and detaches from the plastic carrying the whole adherent
haematon layer.
▲CRITICAL STEP Keep the dish horizontally otherwise the gel polymerizes unevenly.
6) Remove the buffer and replace with 5 mM CaCl2 in 100 mM Tris-HCl.
7) Cut the whole sheet into longitudinal strips using plastic disposable cell scraper to facilitate
removal of alginate sheet from the dish.
8) Cut the hydrogel stripes further and transfer the samples separately into culture wells as above.
Box 3|
In vivo clonal assays: Spleen-Colony-Forming-Unit (CFU-Sd12) and Competitive Marrow
Repopulating Ability (CMRA)
Transfusion
1) Irradiate wild type host mice (C57/Bl6-CD45.2 phenotype; 2-4 month old, 6 to 10 per group) at 9.5
Gy in a γ-irradiator on day-1 of graft injection.
2) Distribute decreasing number of test cells in replicate wells for limiting dilution analysis or sort
presumptive HSCs (i.e. 90, 30 or 10 LinnegSpSK cell per well) from C57/Bl6-CD45.1-ROSA26 mouse in
100 µl IMDM containing 0.5% (vol/vol) BSA. Keep the samples at 4 °C until injection.
3) Add to each donor wells 105 normal competitor C57/Bl6-CD45.2 BM cells in 100 µl IMDM
containing 0.5% (wt/vol) BSA per well.
4) Under mild Avertin anaesthesia (i.e. 10 µg Avertin per 10 g b.w.) inject the totality of cells in 200 µl
medium via the retro-orbital plexus using 30-gauge (0.3x8 mm) needles.
5) Keep the mouse on a 37 °C platform until recovery (≥60 min) then return them for housing.
Spleen removal
6) Anesthetize mice at day 12 after transplantation using 12.5 µg Avertin in 100 µl NaCl per 10 g b.w.
or 1.5% isoflurane inhalation). Shave the left abdominal side with an electric trimmer, immobilize at
the right lateral decubitus position on the dissection pad and then disinfect with Betadine or 70%
(vol/vol) ethanol.
7) Lift the whole skin at the level of spleen with a tweezers then incise the skin and the abdominal
wall with sharp dissecting scissors to obtain a 1 cm long opening. Cover the surrounding area with
sterile, wet gauze.
8) Pull and exteriorize the spleen together with a short segment of intestine using a light tweezers.
9) Ligature separately the lienal and lieno-pancreatic arteries/veins by sterile threads and then cut
these blood vessels carefully to liberate the spleen.
10) Transfer the spleen into 4 ml Zn-7 fixative for 4 h.
▲CRITICAL STEP Surgical removal of the spleen does not compromise marrow regeneration.
CMRA or CRU assay (optional)
11) Following splenectomy return the intestine to the peritoneum, close the wound by two
retractable wound clips and keep the mouse on a 37 °C platform until recovery (30-45 min) for long
term CRU assays49.
Identification of donor-derived (ROSA-26) spleen colonies by X-gal histological staining
(i) Immerse samples in Zn-7 fixative (pH6.7) for 4-24 h (or 4% PAF for 1-2 h) at 4 °C, rinse in 0.9%
(wt/vol) NaCl and then wash 3 times in 100 mM Tris-HCl at pH8.5.
(ii) Incubate samples in Tissue Rinse Solution-A (TRS-A: 2 mM MgCl2, 5 mM EGTA in 100 mM Tris-HCl
(pH8.5) for 1 h at RT, and then in TRS-B: 2 mM MgCl2, 0.01% (wt/vol) Na-deoxycholate, 0.02%
(vol/vol) Nonidet-P40 in 100 mM Tris-HCl (pH8.5) for 30 min at 37 °C.
(iii) Immerse samples in fresh Tissue Stain Solution: TRS-B complemented with 5 mM K3Fe(CN)6, 5
mM K4Fe(CN)6.6H2O and 1 mg ml-1 X-gal (4-chloro-5-bromo-3-indolyl-β-D-galactopyranoside) for 16 h
to 4 d at 37 °C.
(iv) Wash in 100 mM Tris (pH7.4) then post-fix the preparations in Zn-7 for 24 h.
(v) Count uncoloured spleen colonies derived from competitor marrow cells and X-gal positive,
donor-derived blue colonies.
▲CRITICAL STEP Zn-7 is a harmless fixative allowing single or multiple enzyme-histochemical reactions
to perform. Several tissues express endogenous galactosidase which give strong reaction at the
conventional neutral pH but not at higher pH, in contrast to the bacterial galactosidase. Therefore,
working at pH8.5 is critical to preferentially visualise the bacterial enzyme.
? TROUBLESHOOTING Ca2+ strongly inhibits X-gal activity therefore wash the samples extensively after
fixation in Zn-7 which contains 0.05% (wt/vol) Ca-acetate.